Medicament, compositions, and substances for treating and identifying adenocarcinoma of the lung

ABSTRACT

The invention is based on the finding that in mammalian lungs c-myc acts as a molecular switch, specifically inducing an expression pattern in vivo, which results in prototypical mammalian adenocarcinoma of the lung and liver metastasis. A set of factors essential for the processes of tumorigenesis and tumor progression, i.e. cell cycle and apoptosis, cell growth, extracellular signaling, angiogenesis and invasion, is identified, whose expression is significantly changed. In particular, the expression pattern found uncovers the network of molecules leading to mammalian papillary adenocarcinomas of the lung.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to PCT international patent application no. PCT/EP2009/002441, filed on Mar. 30, 2009, claiming priority to European application no. 08075243.9, filed on Mar. 28, 2008. Those applications are incorporated by reference herein.

INCORPORATION BY REFERENCE OF ASCII TEXT FILE

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BACKGROUND OF THE INVENTION

1. Field of the Invention

Embodiments of the invention relate to a medicament, compositions, and substances directed to novel c-myc modulated factors, and the use of said compositions and substances for labelling, identifying, and treating adenocarcinoma of the lung, in particular papillary adenocarcinoma of the lung. Areas of application are the life sciences: biology, biochemistry, biotechnology, medicine and medical technology.

2. Background of the Related Art

The most common cause of cancer-related death in industrial countries is lung cancer. Lung carcinoma is a leading cause of cancer morbidity with tumor entities being classified as small-cell (SCLC) or non-small-cell lung carcinomas (NSCLC). It is generally accepted that several genetic changes appear to be necessary for malignant transformation and these include, amongst others, overexpression of protooncogenes and loss of tumor-suppressor functions. However, despite intense research the molecular causes of lung adenocarcinoma are poorly understood. Nonetheless, there is evidence for the c-Myc protooncogene to play a central role in various types of human tumors.

However, the molecular and cellular mechanisms of carcinogenesis induced by c-myc in vivo are poorly understood, so far. In this regard, the identification of downstream target genes, which distinguish between physiologic and tumorigenic functions of c-Myc in vivo would allow novel approaches for the treatment, diagnosis, and study of cancer.

BRIEF SUMMARY OF THE INVENTION

It would be desirable to make available a medicament and medication, compositions, and substances directed to novel c-myc modulated factors, and the use of said compositions and substances for labelling, identifying, and treating adenocarcinoma of the lung, in particular papillary adenocarcinoma of the lung.

Embodiments of the invention are based on the surprising finding that in mammalian lungs c-myc acts as a molecular switch, specifically inducing an expression pattern in vivo, which results in prototypical mammalian adenocarcinoma of the lung and liver metastasis. A set of factors essential for the processes of tumorigenesis and tumor progression, i.e. cell cycle and apoptosis, cell growth, extracellular signaling, angiogenesis and invasion, is identified, whose expression is significantly changed. In particular, the expression pattern found uncovers the network of molecules leading to mammalian papillary adenocarcinomas of the lung.

We make available a medicament, compositions, and substances directed to novel c-myc modulated factors, and the use of said compositions and substances for labelling, identifying, and treating adenocarcinoma of the lung, in particular papillary adenocarcinoma of the lung.

Embodiments concern a medicament for the treatment of lung carcinoma comprising a composition that decreases the expression or activity of a c-myc modulated gene selected from the group consisting of Prc1, Klt4, Ect2, Cdc20, Stk6, Nek6, Ect2, Birc5, Hspa9a, Cideb Pglyrp, Zfp239, Elf5, Uck2, Smarcc1, Arg1, Hk1, Gapd, Suclg2, Tpi, Gnpnat1, Pign, Gapd, Mre11a, Top2a, Ard1, Hmgb2, Xrcc5, Rrm1, Rrm2, Smarcc1, Npm3, Nol5, Lamr1, H1fx, Lmnb1, Spnr, Npm3, Nola1, Mki67ip, Ppan, Rnac, Grwd1, Srr, Pycs, Pcbd, Mrps5, Lamr1, Mrp112, Rp144, Eif2b, Tomm40, Slc15a2, Slc4a7, Slc4a4, Rangnrf, Kpnb3, Ipo4, Mlp, Stk39, Rbp1, Reck, Areg, Ros1, Arhu, Frat2, Traf4, Myc, Frat2, Cldn2, Gjb3, Gja1, Kra1-18, Col15a1, Dsg2, Ect2, Lcn2, Kng, Hgfac, Adora2b, Spint1, Adam19, Hpn and/or increases the expression or activity of a c-myc modulated gene selected from the group consisting of Cdkn2d, Lats2, Hey1, Stat1, Bnip2, Caipn2, Anp32a, Madh6, Foxf1a, Tbx3, Tcf21, Gata3, Sox2, Crap, Trim30, Klf7, Sox17, Sox18, Meis1, Foxf2, Satb1, Anp32a, Bmp6, Tgfb1, Dpt, Acvrl1, Eng, Zfhx1a, Igfbp5, Igfbp6, Igfbp4, Socs2, Nfkbia, Sox7, Ptpre, Ptpns1, Rassf5, Fkbp7, Sema3f, Vsnl1, Reck, Capn2, Cdh5, Spock2, Thbd, Tie1, Icam2, Tek, Nes, Vwf, Xlkd1, Sparcl1, Marcks, Tenc1, Pcdha6, Lama4, Lama3, Pcdha4, Vtn, Vcam1, Tna, Stab1, Cldn5, Pmp22, Ptprb, Ptprg, Slfn2, Ndr2, Ets1, Sipa1, Ndn, Meox2, Rbp1, Sema7a, Sema3c, Sema3e, Tagln, and Ablim1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Histology of lung tumors of SPC/c-Myc-transgenic mice. Normal subpleural parenchyma of nontransgenic controls (a). Invasive papillary lung adenocarcinoma (PLAC) with maximal diameter of 220 μm besides foci of dispersed BAC: Real papilla with their own stroma (b). Advanced PLAC with folded papillary structures of secondary and tertiary degree (arrow=mitosos) (c). Liver metastasis of PLAC (d).

FIG. 2: Differently expressed genes in lung adenocarcinoma of c-Myc-transgenic mice: known responsiveness/interaction with c-Myc; proportion (%) of genes containing potential c-Myc-binding sites in promoter region

1—known c-Myc-targets and their relatives 2—known c-Myc-targets and their relatives containing potential c-Myc binding sites 3—known c-Myc-responsive genes and their relatives containing potential c-Myc binding sites 4—known c-Myc-responsive genes and relatives 5—new c-Myc-responsive genes containing potential c-Myc binding sites 6—new c-Myc-responsive genes

FIG. 3: RT-PCRs for selected genes: lanes 1-4: control lung; 5-7: pools of small size tumors; 8-12: middle size tumors; 13-15: large size tumors

FIG. 4: Western blot analysis for selected genes: C1-C-3-control non-transgenic lung; T1-T3-papillary lung adenocarcinomas of SPC/c-Myc transgenic mice

FIG. 5: Structure of the transgene (Ehrhardt et al., 2001): SPC-Promoter, human surfactant protein promoter; α1AT, first exon of the non coding alpha 1 antitrypsin gene; I1, intron 1 of the alpha 1 antitrypsin gene fused to the first intron of the c-myc protooncogene; I2, intron 2 of the c-myc protooncogene; SVA, SV40 Poly A signal. Primer binding sites within the transgene are indicated (black boxes); fp, forward primer; rp, reverse primer (a). PCR analysis of SP-C/myc from tail biopsies to identify transgenic mice. Lane 1-2: transgenic mice; Lane 2: amplified fragment of the transgene was digested with Sal 1 to obtain fragments of about 200 and 500 Bp; Lane 3: non transgenic mouse; M, molecular weight standard (b).

FIG. 6: Up regulated genes involved in cell growth in c-Myc-induced lung adenocarcinoma: known responsiveness/binding to c-Myc; presence of c-Myc-binding sites in promoter region

1—known c-Myc-targets and their relatives containing potential c-Myc binding sites 2—known c-Myc-targets and their relatives 3—known c-Myc-responsive genes and their relatives containing potential c-Myc binding sites 4—known c-Myc-responsive genes and relatives 5—new c-Myc-responsive genes containing potential c-Myc binding sites 6—new c-Myc-responsive genes

FIG. 7: RT-PCRs for selected genes: lanes 1-4: control lung; 5-7: pools of small-sized tumors; 8-12: middle-sized tumors; 13-15: large-sized tumors

FIG. 8: Western analysis for selected genes: C1-C-3-control non-transgenic lung; T1-T2-lung adenocarcinomas of SPC/c-Myc transgenic mice

FIG. 9: RT-PCRs for selected genes: lanes 1-4: control lung; 5-7: pools of small size tumors; 8-12: middle size tumors; 13-15: large size tumors

FIG. 10: Western analysis for selected genes: C1-C-3-control non-transgenic lung; T1-T2 lung adenocarcinomas of SPC/c-Myc transgenic mice

FIG. 11: Overview of pathways with involved genes deregulated in c-Myc induced lung adenocarcinoma

FIG. 12: Histology of lung tumors of SPC/c-Myc-transgenic lungs

Normal subpleural parenchyma of non transgenic animals (a). Dispersed and immediately subpleural densely packed BAC: hyperchromatic low columnar lining cells of the alveoli (b). Initial invasive papillary lung adenocarcinoma (PLAC) besides foci of dispersed BAC (c)

FIG. 13: Hierarchical gene clustering. different gene expression regulation in BAC and PLAC.

BAC F—BAC, females BAC M—BAC, males PLAC S—small-sized PLACs PLAC M—middlel-sized PLACs PLAC L—large-sized PLACs

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, embodiments of the invention are directed to the use of at least one biomarker selected from the group consisting of Satb1, Hspa9a, Hey1, Gas1, Bnip2, Capn2, Anp32a, Ddit3, Ccnb2, Cdkn2d (p19), Prc1, Uck2, Srm, Shmt1, Slc19a1, Npm1, Npm3, Nol5, Lamr1/Rpsa, Arhu(Rhou), Traf4, Adam19, Bmp6, Rbp1, Reck, Ect2 (Group A) in the diagnosis of cancer and/or the prognosis of cancer and/or the treatment monitoring of cancer, in particular of lung cancer such as lung adenocarcinoma(s) may be.

For different purposes, it is preferred if the at least one biomarker is a combination of

(a) at least one biomarker selected from the group consisting of Group A, and

(b) the epithelial cell adhesion molecule (EpCAM).

Preferentially, a combination of Satb1 and at least one further biomarker selected from the group of Hspa9a, Hey1, Gas1, Bnip2, Capn2, Anp32a, Ddit3, Ccnb2, Cdkn2d (p19), Prc1, Uck2, Srm, Shmt1, Slc19a1, Npm1, Npm3, Nol5, Lamr1/Rpsa, Arhu(Rhou), Traf4, Adam19, Bmp6, Rbp1, Reck, Ect2, EpCAM is used as the at least one biomarker. Preferably, an appropriate amount of the at least one biomarker is used, in particular an amount for manufacturing a reference, more particular for manufacturing a reference comprising a reference level of said at least one biomarker, such as the level of said at least one biomarker in a sample of a normal healthy individual or the level of a said at least one biomarker in a sample of a patient suffering from cancer may be.

In particular, the at least one biomarker selected from the group consisting of Group A, or the combination of (a) at least one biomarker selected from the group consisting of Group A and (b) the epithelial cell adhesion molecule (EpCAM), is used for monitoring the therapeutic treatment of a patient, wherein the patient is preferably a human being or a transgenic c-myc mouse, suffering from lung cancer, in particular for monitoring the treatment of said patient with irinotecan, paclitaxel and/or 5-fluorouracil and/or the treatment with a drug binding to the epithelial cell adhesion molecule (EpCAM), such as an anti EpCAM antibody may be.

Preferably, at least one biomarker is selected from the group consisting of Ccnb2, Slc19a1, Uck2, Srm1, Nol5a, Arhu, Adam19, Ect2, Shmt1 (Group B) or from the group consisting of Gas1, Bmp6, Bnip2,_Capn2, Ddit3, Hey1 (Group C), and wherein the at least one biomarker preferably further comprises EpCAM, is used, in particular for the diagnosis, prognosis and/or treatment monitoring of PLAC. More preferably, at least one biomarker is selected from the group consisting of Group B and at least one biomarker selected from the group consisting of Group C are used, and wherein said at least two biomarkers preferably further comprise EpCAM.

Another aspect of the invention is directed to a method for diagnosing cancer and/or prognosing cancer and/or staging cancer and/or monitoring the treatment of cancer, in particular lung cancer, such as lung adenocarcinoma(s) may be, comprising the steps of:

(a) measuring the level of at least one biomarker selected from the group consisting of Group A, and wherein said at least one biomarker preferably further comprises EpCAM, in a patient or in a sample of a patient, wherein the patient is preferably a human being or a transgenic c-myc mouse, suffering from or being susceptible to cancer, and

(b) comparing the level of said at least one biomarker in said patient or in said sample to a reference level of said at least one biomarker.

Preferably, the method for diagnosing, prognosing and/or staging cancer and/or monitoring the treatment of cancer, is implemented for monitoring the therapeutic treatment of a patient suffering from lung cancer, in particular the treatment with irinotecan, paclitaxel and/or 5-fluorouracil and/or the treatment with a drug binding to the epithelial cell adhesion molecule (EpCAM), such as an anti EpCAM antibody may be.

In a particularly preferred embodiment, at least one biomarker is selected from the group consisting of Group B or from the group consisting of Group C, and wherein said at least one biomarker preferably further comprises EpCAM, in particular for the diagnosis, prognosis, staging and/or treatment monitoring of PLAC.

In particular, the method is preferably implemented to distinguish between different subtypes of lung cancer, such as (but not limited to) lung adenocarcinomas as defined by BAC or PLAC, wherein a significantly increased level of Group B in comparison with the level of a normal individual or a significantly decreased level of Group C in comparison with the level of a normal individual is indicative of PLAC.

More particularly, at least one biomarker selected from the group consisting of Group B and at least one biomarker selected from the group consisting of Group C are measured to distinguish between different subtypes of lung cancer, such as (but not limited to) lung adenocarcinomas as defined by BAC or PLAC.

A significantly increased level of Group B in comparison with the level of a normal individual and a significantly decreased level of Group C in comparison with the level of a normal individual is then indicative of PLAC.

According to embodiments of the invention, the medicament or medication for the treatment of lung carcinoma, in particular of papillary adenocarcinomas of the lung, comprises a pharmaceutically effective amount of a composition that decreases the expression or activity of a c-myc modulated gene selected from the group consisting of Prc1, Klt4, Ect2, Cdc20, Stk6, Nek6, Ect2, Birc5, Hspa9a, Cideb Pglyrp, Zfp239, Elf5, Uck2, Smarcc1, Hk1, Gapd, Suclg2, Tpi, Gnpnat1, Pign, Gapd, Mre11a, Top2a, Ard1, Hmgb2, Xrcc5, Rrm1, Rrm2, Smarcc1, Npm3, Nol5, Lamr1, H1fx, Lmnb1, Spnr, Npm3, Nola1, Mki67ip, Ppan, Rnac, Grwd1, Srr, Pycs, Pcbd, Mrps5, Lamr1, Mrp112, Rp144, Eif2b, Tomm40, Slc15a2, Slc4a7, Slc4a4, Rangnrf, Kpnb3, Ipo4, Mlp, Stk39, Rbp1, Reck, Areg, Ros1, Arhu, Frat2, Traf4, Myc, Frat2, Cldn2, Gjb3, Gja1, Krt1-18, Col15a1, Dsg2, Ect2, Lcn2, Kng, Hgfac, Adora2b, Spint1, Adam19, Hpn (Group D) and/or increases the expression or activity of a c-myc modulated gene selected from the group consisting of Cdkn2d, Lats2, Hey1, Stat1, Bnip2, Capn2, Anp32a, Madh6, Foxf1a, Tbx3, Tcf21, Gata3, Sox2, Crap, Trim30, Klf7, Sox17, Sox18, Meis1, Foxf2, Satb1, Anp32a, Bmp6, Tgfb1, Dpt, Acvrl1, Eng, Zfhx1a, Igfbp5, Igfbp6, Igfbp4, Socs2, Nfkbia, Sox7, Ptpre, Ptpns1, Rassf5, Fkbp7, Sema3f, Vsnl1, Reck, Capn2, Cdh5, Spock2, Thbd, Tie1, Icam2, Tek, Nes, Vwf, Xlkd1, Sparcl1, Marcks, Tenc1, Pcdha6, Lama4, Lama3, Pcdha4, Vtn, Vcam1, Tna, Stab1, Cldn5, Pmp22, Ptprb, Ptprg, Slfn2, Ndr2, Ets1, Sipa1, Ndn, Meox2, Rbp1, Sema7a, Sema3c, Sema3e, Tagln, and Ablim1 (Group E).

Preferably, the medicament or medication further comprises a pharmaceutically acceptable carrier and/or recipient and/or diluent. In particular, the medicament or medication for the treatment of lung carcinoma comprises a composition that increases and/or decreases the expression or activity a c-myc modulated gene in a mammal, preferably in a human being or a mouse, more preferably in a human being.

The genes according to embodiments of the invention concern genes of mammalia, preferably genes of the genome of mus musculus or homo sapiens, in particular the respective genes of homo sapiens are preferred. Within the context of the invention, it is in particular preferred if the c-myc modulated gene is selected from the group of genes being grayscaled in the Tables 2, 4 and 6 or preferably Table 6. Preferably, the medicament or medication according to the invention comprises a pharmaceutically effective amount of a composition as detailed below.

Within the context of the present invention an antisense composition is provided, wherein the antisense composition comprises a nucleotide sequence complementary to a coding sequence of a c-myc modulated gene selected from the group consisting of Group D.

In this regard, the term “coding sequence” is directed to the portion of an mRNA which actually codes for a protein. The term “nucleotide sequence complementary to a coding sequence” in particular is directed to an oligonucleotide compound, preferably RNA or DNA, more preferably DNA, which is complementary to a portion of an mRNA, and which hybridizes to and prevents translation of the mRNA. Preferably, the antisense DNA is complementary to the 5′ regulatory sequence or the 5′ portion of the coding sequence of said mRNA.

It is preferred that the antisense composition comprises a nucleotide sequence containing between 10-40 nucleotides, preferably 12 to 25 nucleotides, and having a base sequence effective to hybridize to a region of processed or preprocessed mammalian, preferably human or murine, more preferably human mRNA.

In particular, the composition comprises a nucleotide sequence effective to form a base-paired heteroduplex structure composed of mammalian, preferably human or murine, more preferably human RNA transcript and the oligonucleotide compound, whereby this structure is characterized by a Tm of dissociation of at least 45° C.

Further, a siRNA composition is provided, wherein the siRNA composition comprises an siRNA reducing or preferably inhibiting the expression of a c-myc modulated gene selected from Group D. Embodiments may employ siRNA for use in modulating the level of protein presence in the cell. SiRNA oligonucleotides directed to said genes specifically hybridize nucleic acids encoding the gene products of said genes and interfere with gene expression of said genes.

Preferably, the siRNA composition comprises siRNA (double stranded RNA) that corresponds to the nucleic acid ORF sequence of the gene product coded by one of said mammalian, preferably human or murine, more preferably human genes or a subsequence thereof; wherein the subsequence is 19, 20, 21, 22, 23, 24, or 25 contiguous RNA nucleotides in length and contains sequences that are complementary and non-complementary to at least a portion of the mRNA coding sequence.

Nucleotide sequences and siRNA may be prepared by any standard method for producing a nucleotide sequence or siRNA, such as by recombinant methods, in particular synthetic nucleotide sequences and siRNA is preferred.

Furthermore, an antibody composition is provided, wherein the antibody composition comprises a pharmaceutically effective amount of an antibody or fragment thereof that binds to a polypeptide encoded by a gene selected from the group consisting of Group D.

Within the inventive context, antibodies are understood to include monoclonal antibodies and polyclonal antibodies and antibody fragments (e.g., Fab, and F(ab′)₂) specific for one of said polypeptides. Polyclonal antibodies against selected antigens may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, various fowl, rabbits, mice, or rats. Preferably, monoclonal antibodies are used in the antibody compositions of the invention which may be readily generated using conventional techniques (see Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are incorporated herein by reference).

In particular, antibodies or fragments thereof specific for the gene products involved in extracellular signaling, angiogenesis and invasion, in particular specific for the gene products of Rbp1, Reck, Areg, Ros1, Arhu, Frat2, Traf4, Myc, Frat2, Cldn2, Gjb3, Gja1, Krt1-18, Col15a1, Dsg2, Ect2, Lcn2, Kng, Hgfac, Adora2b, Spint1, Hpn, more particular for said gene products highlighted in Table 3, are preferred.

Further, a polypeptide composition (also termed as polypeptide composition (1)) is provided, wherein the polypeptide composition comprises a polypeptide coded by the sequence of a c-myc modulated gene selected from the group consisting of Group E.

Preferably, the polypeptide composition comprises a peptide coded by the entire ORF/coding sequence of one of said genes. Such peptides include isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, in particular 100% identity, to the entire amino acid sequence of the gene product of one of said genes.

In particular, a polypeptide composition comprising a polypeptide involved in extracellular signaling, angiogenesis and invasion is preferred. Polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

Furthermore, a vaccine composition is provided comprising a polypeptide coded by the sequence of a c-myc modulated gene selected from the group consisting of Group D. The vaccine composition for immunogenic and vaccine purposes in eliciting lung adenocarcinoma-specific immune responses preferably provides polypeptides comprising antigenic determinants from said gene products. An antigenic determinant of the gene product of one of said genes can be provided either by the full sequence of the gene product concerned, or by such subsequences as may be desired, e.g. a sequence fragment comprising at least 25%, e.g. at least 50% or 75% of the full sequence of the gene product concerned, e.g. a N-terminal or C-terminal sequence fragment.

In particular, a vaccine composition is preferred that comprises an antigenic determinant of a polypeptide involved in extracellular signaling, angiogenesis and invasion. A further aspect of the invention concerns the use of a composition that decreases the expression or activity of a c-myc modulated gene selected from the group consisting of Group D and/or increases the expression or activity of a c-myc modulated gene selected from the group consisting of Group E for the preparation of a medicament or medication, preferably for the preparation of a medicament or medication for preventing, treating, or ameliorating lung carcinoma, more preferably for preventing, treating, or ameliorating adenocarcinoma of the lung, in particular papillary adenocarcinomas of the lung.

In particular, an antisense composition, an siRNA composition, an antibody composition or the polypeptide composition (1) as detailed herein, or a combination thereof, is used for the preparation of the medicament or medication. In one embodiment, a nucleotide composition (also termed as nucleotide composition (1)) is used for the preparation of said medicament or medication, wherein the nucleotide composition comprises a nucleotide sequence of a c-myc modulated gene selected from the group consisting of Group E.

The nucleotide composition particularly comprises a nucleic acid being from about 20 base pairs to about 100,000 base pairs in length, wherein the nucleotide sequence is included. Preferably the nucleic acid is from about 50 base pairs to about 50,000 base pairs in length. More preferably the nucleic acid is from about 50 base pairs to about 10,000 base pairs in length. Most preferred is a nucleic acid from about 50 pairs to about 4,000 base pairs in length. The nucleotide sequence can be a gene or gene fragment that encodes a protein, an oligopeptide or a peptide. Preferably, the nucleotide sequence of the present invention may comprise a DNA construct capable of generating a gene product coded by one of said genes and may further include an active constitutive or inducible promoter sequence.

In particular the nucleotide composition comprises a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to the amino acid sequence of the gene product of one of said genes. In this regard, nucleotide sequences coding for polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more preferred, and those with at least 99% identity are most preferred. In particular, it is preferred if the nucleotide sequence encodes a polypeptide with 100% identity to the entire amino acid sequence of the gene product of one of said genes.

In particular, the nucleotide composition comprises a DNA sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to the ORF (or coding sequence, respectively) of one of said genes over the entire coding region. In this regard, nucleotide sequences which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred. In particular, it is preferred if the nucleotide sequence encodes a DNA sequence that has 100% identity to the entire ORF of one of said genes over the entire coding region.

The nucleotide composition may further comprise an enhancer element and/or a promoter located 5′ to and controlling the expression of said therapeutic nucleotide sequence or gene. The promoter is a DNA segment that contains a DNA sequence that controls the expression of a gene located 3′ or downstream of the promoter. The promoter is the DNA sequence to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene, typically located 3′ of the promoter.

Preferably, a pharmaceutically effective amount of one of the compositions specified above or a pharmaceutically effective amount of a combination thereof is used, in particular furthermore a pharmaceutically acceptable carrier and/or recipient and/or diluent is used for the preparation of the medicament or medication. In a further aspect of the invention, an immunogenically effective amount of the vaccine composition as detailed herein is used for the preparation of a vaccine, wherein preferably furthermore an adjuvant is used. In particular, it is preferred if the vaccine composition and the adjuvant are admixed with a pharmaceutically effective vehicle (excipient).

Yet another aspect of the invention concerns the use of novel c-myc modulated genes and/or gene products of thereof to screen for and to identify drugs against lung carcinoma, preferably against adenocarcinoma of the lung, more preferably against papillary adenocarcinoma of the lung, wherein at least one or more genes and/or one or more gene products of thereof selected from the group of Group D and/or Group E is used.

In one embodiment, a nucleotide composition (termed as nucleotide composition (2), respectively) is used as the one or more genes, wherein the nucleotide composition comprises a nucleotide sequence of a c-myc modulated gene selected from the group consisting of Group D.

Said nucleotide composition particularly comprises a nucleic acid being from about 20 base pairs to about 100,000 base pairs in length, wherein the nucleotide sequence is included. Preferably the nucleic acid is from about 50 base pairs to about 50,000 base pairs in length. More preferably the nucleic acid is from about 50 base pairs to about 10,000 base pairs in length. Most preferred is a nucleic acid from about 50 pairs to about 4,000 base pairs in length. The nucleotide sequence can be a gene or gene fragment that encodes a protein, an oligopeptide or a peptide. Preferably, the nucleotide sequence of the present invention may comprise a DNA construct capable of generating a gene product coded by one of said genes and may further include an active constitutive or inducible promoter sequence.

Preferably, this nucleotide composition comprises a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to the amino acid sequence of the gene product of one of said genes. In this regard, nucleotide sequences coding for polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more preferred, and those with at least 99% identity are most preferred. In particular, it is preferred if the nucleotide sequence encodes a polypeptide with 100% identity to the entire amino acid sequence of the gene product of one of said genes.

In particular, the nucleotide composition (2) comprises a DNA sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to the ORF (or coding sequence, respectively) of one of said genes over the entire coding region. In this regard, nucleotide sequences which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred. In particular, it is preferred if the nucleotide sequence encodes a DNA sequence that has 100% identity to the entire ORF of one of said genes over the entire coding region.

In another embodiment, a polypeptide composition (also termed as polypeptide composition (2)) is used as the one or more gene products, wherein the polypeptide composition comprises a polypeptide coded by the sequence of a c-myc modulated gene selected from the group consisting of Group D.

Such peptides include isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the entire amino acid sequence of the gene product of one of said genes. Preferably, the polypeptide composition comprises a peptide coded by the entire ORF/coding sequence of one of said genes. In particular, a polypeptide composition comprising a polypeptide involved in extracellular signaling, angiogenesis and invasion, as coded by Rbp1, Reck, Areg, Ros1, Arhu, Frat2, Traf4, Myc, Frat2, Cldn2, Gjb3, Gja1, Kra1-18, Col15a1, Dsg2, Ect2, Lcn2, Kng, Hgfac, Adora2b, Spint1, Adam19, Hpn is preferred.

Preferably, one or more of said genes and/or their gene products, in particular the nucleotide composition (2) and/or the polypeptide composition (2), is incubated with a compound to be tested and changes in the expression of said genes and/or derived sequences and/or the function of said gene products are determined. In particular, the antisense composition, the siRNA composition, the antibody composition, the nucleotide composition (1) or the polypeptide composition (1), as detailed herein, or a combination thereof, is used as test compound.

More preferably, said use for screening and identifying drugs comprises the steps of: (1) contacting a test cell expressing, preferably overexpressing, at least one of said genes with a test compound; (2) detecting the expression level of said gene; and (3) determining the compound that suppresses said expression level compared to a normal control level of said gene as an inhibitor of said gene.

In particular, a compound that enhances the expression or activity of one of said genes is identified by the inventive use comprising the steps of: (1) contacting a test cell expressing, in particular overexpressing, said non-small cell lung cancer-associated gene with a test compound; (2) detecting the expression level of said non-small cell lung cancer-associated gene; and (3) determining the compound that increases said expression level compared to a normal control level of said gene as an enhancer of said non-small cell lung cancer-associated gene. Within this context, it is particularly preferred that the use comprises the steps of: (1) contacting a test compound with a polypeptide encoded by the selected gene; (2) detecting the binding activity between the polypeptide and the test compound; and (3) selecting a compound that binds to the polypeptide.

More specifically, said use comprises the steps of (a) contacting a test compound with a polypeptide encoded by the selected gene; (b) detecting the biological activity of the polypeptide of step (a); and (c) selecting a compound that suppresses the biological activity of the polypeptide encoded by the gene selected from the group consisting of Group D in comparison with the biological activity detected in the absence of the test compound, and/or enhances the biological activity of the polypeptide encoded by the polynucleotide selected from the group consisting of Group E in comparison with the biological activity detected in the absence of the test compound. Advantageously, cell proliferation is detected as biological activity, or any other biological activity of the cell related to carcinogenesis or tumorigenesis, in particular the presence of tumor markers known in the art, is detected.

In a further preferred embodiment the use comprises the steps of: (1) contacting a test compound with a cell into which a vector comprising the transcriptional regulatory region of one or more of the selected genes and a reporter gene that is expressed under the control of the transcriptional regulatory region has been introduced; (2) measuring the activity of said reporter gene; and (3) selecting a compound that reduces the expression level of said reporter gene when the selected gene is an up-regulated gene selected from the group consisting of Group D, and/or that enhances the expression level of said reporter gene when the selected gene is a down-regulated gene selected from the group consisting of Group E, as compared to a control.

More particular, the use comprises drugs, in particular a test compound and/or a medicament or medication as specified above, wherein the drugs regulate the expression of one or more of said genes and/or the function of one or more of said gene products and/or their derived molecules, and said drugs are used for the production of means for treating, identifying or labeling adenocarcinoma, in particular papillary adenocarcinoma, of the lung.

In one embodiment of the use for screening and identifying drugs DNA and/or related molecules encoding one or more of said gene products and/or derived structures are used, and/or one or more polypeptides, peptides and/or derived molecules having the function of one or more of said gene products, are used.

Yet another aspect of the invention concerns a procedure for identifying, labelling and/or treating lung carcinoma, in particular adenocarcinoma of the lung, more particular papillary adenocarcinoma of the lung, wherein a biological and/or biotechnological system is contacted with a soluble substance, such as an oligonucleotide sequence or antibody may be, having affinity with at least one of the genes selected from the group of Group D and/or mRNA encoded thereby and/or their gene products and/or parts thereof and wherein the soluble substance is linked with a marker.

Preferably, the biological or biotechnological system is an organism, a tissue, a cell, a part of a cell, a DNA, a RNA, a cDNA, a mRNA, a cRNA, a protein and/or a peptide and/or a derived structure and/or contains the same. In particular, the biological system is a cell derived from a tumor or of a tumor metastasis, preferably a cell derived from a lung tumor or of a liver metastasis thereof or the biotechnological system is an oligonucleotide library derived thereof.

Particularly, the biological or biotechnological system employed for the procedure comprises cells of a tumor malignancy of the lung and/or an oligonucleotide library and/or a protein library and/or a peptide library derived thereof, such as a transgenic animal, in particular a c-myc mouse as described herein, or biological material received thereof or biological material received from a human lung cancer patient may be.

As the, at least partially, soluble substance in particular oligonucleotides, proteins, peptides or structures derived of thereof are suitable. These have the advantage that they can recognize specifically two or three-dimensional target structures on the molecular level. Beyond that, they provide the favourable characteristic that the recognition usually takes place in aqueous physiologically buffered solutions and leads to a specific association/binding with the targeted structure.

In one embodiment of the procedure, a nucleotide sequence according to the antisense composition, an siRNA according to the siRNA composition, an antibody according to the antibody composition, a nucleotide sequence according to the nucleotide composition (1) or a polypeptide according to the polypeptide composition (1), as described herein, or a combination thereof, is used as the soluble substance. In particular, complementary oligonucleotides, such as a primer or primer pairs described herein, is used as the substance having specific affinity.

Thereby, monoclonal and/or polyclonal antibodies and/or antibody fragments are particularly suitable, since they are formed as stable highly specific structures, which are, in principle, producible against all possible molecular target structures. In a favourable embodiment of the procedure human and/or bispecific antibodies or human and/or bispecific antibody fragments are used. In particular, monoclonal antibodies and/or antibody fragments are thereby suitable.

For implementing the invention it is preferred, if the marker according to invention is selected as an element, an isotope, a molecule and/or an ion or is composed of thereof, such as a dye, contrast means, chemotherapeutic agent, radionuclide, toxin, lipid, carbohydrate, biotin, peptide, protein, microparticle, vesicle, polymer, hydrogel, cellular organelle, virus and/or whole cell, in particular if the marker is formed as dye labeled and/or enzyme-labeled secondary antibodies and/or as protein A and/or as protein G or structures derived of thereof.

The linkage between the marker and the substance is favourably chemically, electrostatically and/or over via hydrophobic interactions, such as there is sufficient connection stability for the use of the marked substance for identifying, labelling and treating of metastatic cells, preferably if the linkage is covalent. For the increase of the sensitivity of the procedure according to invention also the simultaneous use of several substances is in particular suitable, in particular if they comprise two or more substances having each affinity with one of the targets or their mRNA sequences or their gene products.

In particular, for a specific recognition/identification substances are preferred, which bind with an affinity above the association constant Ka=1000 M-1 to the target structure. Preferably, the procedure according to the invention comprises contacting the biological or biotechnological system with a plurality of said soluble substances.

For implementing the procedure according to the invention it is favourable to use one of the following methods—PCR, in vitro translation, RT-PCR, gel electrophoresis, Western Blot, Northern Blot, Southern Blot, ELISA, FACS measurement, chromatographic isolation, UV microscopy, immunohistochemistry, screening of solid phase bound molecules or tissues and/or biosensory investigation—whereby by amplification, isolation, immobilization and/or detection and/or by combinations of thereof a particularly simple conversion of the procedure according to invention is made possible for the examined sample, in particular if furthermore a statistic analysis is accomplished.

The procedure according to invention is preferably implemented by using molecules, cells and/or tissue, in particular being immobilized or synthesized on a planar surface, e.g. spatially addressed. on a nitrocellulose or PVDF membrane, or is linked to the cavity of a microtiter/ELISA plate or on the bottom of a cell culture container, in particular on a glass or plastic chip (biochip).

Favourably, as solid phase bound molecules, substances are used, having affinity for at least one, in particular two, preferably three, more preferably four, most preferably five of the selected target genes and/or their variants and/or parts thereof and/or their mRNA and/or their gene products and/or cleavage products derived thereof, polypeptides or peptides.

The identification of the target structures, e.g. of factors in an immobilized section of tissue or of cells of a cell culture, can take place thereby with arbitrarily marked molecules, which are brought on the surface in solution, e.g. (fluorescence marked/labeled) antibodies. If a RT-PCR is accomplished, then favourably appropriate oligonucleotide probes and/or primers are used. For implementing the procedure according to invention immobilized molecule libraries, e.g. DNA or antibody libraries are used, which associate with the target structure(s) in solution, e.g. with a cDNA library, a PCR product library or with cells of a secondary tumor.

Substances bound to the molecule libraries are particularly identified by the use of appropriately marked probes, e.g. dye labeled oligonucleotides. If unabeled primary antibodies are used, preferably labeled secondary antibodies are used for detection. Furthermore, the use of other proteins, e.g. enzymes, or streptavidin or parts of thereof is suitable. For the diagnosis of a tumor, in particular by in vitro and/or in vivo diagnostics, with the help of the procedure according to invention preferably optically (or radiographically) sensitive equipment, is used, e.g. an UV microscope, scanner or ELISA reader, photometer, or szintigrafic equipment, e.g. X-ray gadget are appropriate.

A further aspect of the invention concerns the procedure according to the invention for diagnosing lung cancer, in particular adenocarcinoma of the lung, more particular papillary adenocarcinoma of the lung, or a predisposition to developing lung cancer in a subject, comprising determining an expression level of a c-myc modulated gene selected from the group consisting of Group D, in a biological sample derived from the subject, wherein an increase of said level compared to a normal control level of said gene indicates that said subject suffers from or is at risk of developing lung cancer, in particular if said increase is at least 200% greater than said normal control level, and whereby preferably said level is determined for a plurality of said genes.

Preferably, this procedure further comprises determining an expression level of a c-myc modulated gene selected from the group consisting of Group E in a biological sample derived from the subject, and wherein a decrease in said level compared to a normal control level indicates said subject suffers from or is at risk of developing non-small cell lung cancer, in particular if said decrease is at least 200% lower than said normal control level, and whereby preferably said level is determined for a plurality of said genes.

In practice, the procedure according to the invention preferably comprises any one of the methods: detecting the mRNA of the selected genes, detecting the protein encoded by the selected gene or detecting the biological activity of the protein encoded by the selected gene.

In one embodiment of the procedure it is preferred if the hybridization of a selected gene probe to a gene transcript of a biological sample is determined, in particular if a primer or primer pair, as described herein, is used as gene probe and/or if the hybridization step is carried out on a DNA array.

Another aspect of the invention concerns a method appropriate for the use and/or the procedure according to the invention, in particular for identifying adenocarcinoma of the lung, comprising the steps of isolating a biological sample from biological material received from an organism, in particular from a human patient, suffering from lung cancer or from an organism to be tested for its susceptibility to lung cancer, and determining the level of Group D and/or Group E or of fragments of thereof or of a selection of thereof in the biological sample by screening the presence of said proteins or of fragments of thereof or of mRNA coding for the same.

In one embodiment it is preferred, if the method comprises the steps of isolating a biological sample from biological material received from a healthy organism, in particular from a human being and pairwisely comparing the gene expression profiles determined for the isolated biological samples by correlating the levels measured; and/or a standard is used for determining the changes of expression, in particular the level of gene expression of at least one of the genes to be screened derived from a gene expression profile for a healthy tissue of the organ, wherein the tumor is formed, is used.

In particular, it is preferred, if

-   -   the isolated biological sample is a tissue, a cell, a cellular         compartment, total RNA, total protein or a body fluid; and/or     -   the standard comprises values for the expression levels of the         targeted genes and/or values for β-Actin, in particular being         determined parallely to the gene expression profile (2); and/or     -   the levels of expression of the at least two genes coding for         the factors screened are normalized with respect to the levels         of expression of the respective genes in healthy tissue wherein         the tumor is formed, in particular by using the standard for         normalizing.

Moreover, the gene expression profiling preferably comprises the steps of

-   -   synthesizing a cDNA library derived of the isolated RNA by         RT-PCR,     -   synthesizing a cRNA library, derived of the cDNA library by         second strand cDNA synthesis and in vitro transcription of the         double stranded cDNA,     -   producing a RNA fragment library derived of the cRNA library by         hydrolytic cleavage into RNA fragments,     -   performing a hybridization assay by incubating an         oligonucleotide array including spatially addressed solid phase         bound oligonucleotide sequences coding for the at least two         oligonucleotide sequences to be screened or for parts of thereof         or for sequences being complementary of the same, with the cRNA         fragment library,     -   scanning the hybridization pattern of the oligonucleotide array.

Particularly, the use of labelled ribonucleotides, such as biotin labelled ribonucleotides may be, is preferred for the in vitro transcription or the method wherein antibodies specific for one or more of said targets are used. In particular, it is preferred according to the invention, if microarrays are used for performing the hybridization assays.

Yet another preferable aspect of the invention concerns a testkit for identifying and/or determining mammalian, in particular human and/or murine, preferably human, tumor malignancies, in particular adenocarcinoma of the lung, more particular papillary adenocarcinoma of the lung, comprising a soluble substance as specified above, in particular comprising two or more detection reagents which bind to one or more genes selected from the group consisting of Group D, and/or Group E, and/or mRNA of thereof and/or polypeptides encoded thereby, for a fast and easy implementation of the invention.

Histopathology of Lung Tumors.

Lung specimens of non-transgenic and transgenic mice were examined at the age of 9-13 months. Mostly a medium sized or large solid tumor nodule was found in transgenic lung and such nodule may filled an entire lobe, while the remaining parenchyma appeared to be macroscopically unaffected. Histologically a ubiquitous proliferation of the peripheral pulmonary epithelium was evident, and the solid tumors were only part of it (FIGS. 1 b and 1 c). The alveoli and parts of the terminal bronchioli were lined by hyperchromatic low columnar cells, exhibiting classic lepidic growth pattern. These cells were dispersed throughout the whole lung and eventually formed a confluent monolayer in the alveoli. The lesional cells were all quite similar and showed only slight nuclear polymorphism, but severe nuclear atypia, as defined by vesicular enlargement and prominent nucleoli. Mitosis was repeatedly seen, e.g. at least 1 mitosis/high power field (=HPF) in confluent lesions. There was lepidic growth and severe cellular atypia typical for bronchiolar alveolar carcinoma (BAC). Unlike BAC the macroscopically visible solid tumors were invasive growing carcinomas, all invariably with papillary growth pattern. While initial papillary lung adenocarcinomas (PLAC) were minimal invasive (lesions), advanced PLAC developed the whole range of malignancy as gross bronchial and vascular invasion and finally liver metastasis (FIG. 1 d). A lung lobe might contain up to five tumor foci. By extrapolation one might expect up to 25 multicentric invasive tumors for an entire lung. Note, solid invasive tumors, i.e. PLACs, were taken for gene expression analysis.

Genome Wide Transcript Profiling

To better understand the transforming capacity of over expressed c-Myc, gene expression profiles in c-Myc-induced PLACS of transgenic mice with histologically confirmed normal lung of non transgenic animals were compared. To identify key transcriptional events associated with malignant growth RNA was prepared from tumor sets of small, middle and large size (see Methods) and analyzed by oligonucleotide microarrays.

Specifically, c-Myc transcript level was more than 50 fold induced in tumor tissues of transgenic mice when compared with lung tissues of normal non-transgenic mice Induction of c-Myc protein expression was confirmed by Western blot analysis (FIG. 4). The expression of c-Myc heterodimeric partner Max was, however, only slightly increased (FC 1,21-1,55 in various tumor samples). The gene coding for Mad4, which competes with c-Myc for Max binding to represses c-Myc activity was expressed in all lung tissues at the same level. Furthermore, Miz-1, which is one of the possible mediator of c-Myc induced gene (Oster et al., 2002), was abundantly expressed in all lung tissues without differences in expression between tumor and control samples. Thus, c-Myc partners were not substantially regulated in response to c-Myc over expression.

Statistical analysis of replicates with T-test and more stringent comparative ranking was performed to identify significantly induced and repressed genes in tumor sets in comparison with non transgenic lungs (see Methods). Array analyses yielded 162 genes (not functionally characterized ESTs were not taken into consideration) with significantly increased expression levels (mean FC >3, p-value in T-test <0,05 and 100% of “Increase” calls in comparative ranking analyses in one or more sets of tumors—see Methods for more details) and about twice as many genes (301) were identified with repressed expression (FC <−3, p-value in T-test <0,05 and 100% “Decrease” accordingly) in c-Myc-induced lung tumors (Table 1). Then, genes differently expressed in lung tumors with published data using the Myc Target Gene database were compared. This database provides valuable information on Myc-responsive genes for various cell types and species (Zeller et al., 2003; Zeller et al., 2003; Zeller et al., 2003). Known c-Myc-responsive genes were identified, i.e. genes whose expression was changed in response to c-Myc activation as well as known c-Myc targets, e.g. genes known to bind c-Myc directly. The results are included in Table 1 and Table 2. From 162 up regulated genes in tumors 29% proved to be previously reported as c-Myc-targets (designated as “T” in the Table 2) or their relatives (“rT”), i.e. these belong to gene families, which include known c-Myc-target (FIG. 2). As expected, many of these genes coded for proteins involved in cell cycle regulation (Cdk4, Ccnb1, Cks2) and apoptosis (Hspa9a, Trp53). Additional 14,8% were known c-Myc responsive genes (“R”) or their relatives (“rR”) which included, amongst others, Tfdp1 and cdc2a, involved in cell cycle regulation (Table 2a). Among the 301 repressed genes identified in tumors it only 7% were found to be known c-Myc-targets or their relatives and additional 13,6% reported as c-Myc responsive genes or the relatives (Table 1). Thus, much more up regulated genes found in the study, have been previously reported to interact directly with c-Myc when compared with repressed genes. About half of the genes with increased expression in papillary lung adenocarcinomas (56,2%) and the majority of down regulated genes (79,4%) were not previously described as c-Myc-responsive. Thus, by use of the genetic disease model it was possible to identify novel c-Myc-responsive genes and these included over expressed Stk6, Nek6, Prc1, Ect2, Birc5 and the repressed Cdkn2d, Lats2, Bnip2, Hey1, all of which are highly interesting disease candidate genes for their essential role in the regulation of cell cycle and apoptosis. Some genes known as c-Myc responsive genes (cEBPalpa, cyclinD1 etc.) were altered in lung adenocarcinomas in opposite direction when compared with previously reported studies which points to cell-type specific responses to c-Myc. These genes are annotated in the Table 2a,b as cursive.

To identify genes which may be directly regulated by c-Myc a computational tool was applied to search for potential c-Myc-binding sites in promoter regions of differently regulated genes. A specific set of mononunucleotide weight matrices with E-box as a core sequence available in TRANSFAC® professional database (see Methods) was applied. The results of this search are included in Table 2a,b and summarized in Table 1 and FIG. 2. With the stringent cut-offs used much more potential c-Myc binding sites in promoters of genes with enhanced expression were found compared with repressed genes (Table 1), with the highest score in the known c-Myc targets (e.g. Cdk4, Kif11, Hspa9a, Fasn, Shtm1, Srm, Apex1 and others). A number of new putative gene targets containing c-Myc-binding sites in their promoters like Prc1, Elf5, Klf7 and others (Table 2a) was also identified Notable, in lung adenocarcinomas repression of several transcription factors was detected, some of them carrying potential c-Myc-binding sites in their promoters, e.g. Foxf1, Tbx3, KLf7 (Table 2b).

The functional analysis of regulated genes in c-Myc induced lung adenocarcinomas indicated that they code for proteins involved in a broad spectrum of cell metabolic and signalling pathways. In the first part of the study genes were described which are associated with cell cycle regulation and intracellular control for cell division as well as those coding for transcription factors. The genes are compiled in Table 2a, b and known c-Myc responsive genes and c-Myc targets are indicated (see above for definition). In addition the number of identified c-Myc-binding sites in promoter regions is also included. Additionally, RT-PCR was performed for 9 genes involved in cell cycle regulation and apoptosis to verify their regulation in tumors by an alternative method The changes in gene expression found in lung tumors by both methods were in good agreement (FIG. 3, Table 3). Moreover, Western blot analysis for two selected genes Nek6 and Hspa9a (Hsp70) confirmed their over expression in tumors at the protein level (FIG. 4).

No qualitative differences in gene expression profiles in relation to tumor size were observed. This agrees with the histological examination where all tumors were classified as papillary adenocarcinomas of various sizes. However, some quantitative differences (up to 2-3 fold) in expression level of several genes between small and large tumors were observed. Among those genes whose expression vel was substantially higher in small size tumors were members involved in cell cycle regulation and included the proto-oncogene ect2. Evidently, this underlines their important role in the onset of tumor growth. Furthermore, the expression of some genes was not detected in tumor samples while their expression was abundant in non-transgenic healthy control lungs (highlighted by grey row accordingly in Table 2b). These included, among others, pro-apoptotic Ddit3 and transcription factor Nr2f1 which exerts anti-AP-1 activity and is a mediator of anti-cancer effect of retinoic acid (Lin et al., 2002).

Thus, in this first study on the oncogenomics of c-Myc-induced lung tumours activation of Myc-target genes was identified greatly contributing to the promotion of cell cycle as well as activation of genes involved in inhibition of apoptosis, therefore, providing a selective advantage for tumor growth, as discussed below.

Genome-wide expression of transcripts in solid tumors induced by targeted c-Myc-expression in alveolar epithelial cells was studied. Note, these tumors were classified as papillary lung adenocarcinomas. To follow the dynamic changes in the gene expression during carcinogenesis, sets of small-, middle- and large-sized tumors were examined separately. By use of the mouse whole genome microarrays it was possible to investigate expression of more than 12480 genes in c-Myc-induced lung tumors and compared findings with normal non-transgenic lungs. Stringent criteria were applied to determine significantly regulated genes in tumors (see “Material and Methods”). As shown in Table 4, many of these genes coded for cell metabolism, DNA and RNA synthesis, ribosomal biogenesis, protein synthesis and transport. Additionally, regulated genes were subjected to bioinformatic analysis and for comparison with findings deposited in a publicly available database termed the Myc Target Gene. The results are included in the Table 4 and summarized in FIG. 6. Remarkably, about one third of the genes involved in meolism and growth proved to be known as c-Myc-targets (designated as “T” in the Table 4) or their relatives (“rT”), i.e. belong to gene families, whose members have been shown in the past to bind c-Myc directly. Amongst them thymidilate synthase, thymidine kinase, fatty acid synthase, lactate dehydrogenase1A, spermidine synthase, nucleolin, nucleophosmin, polyA binding protein 4, several ribosomal proteins, eukaryotic translation initiation factor 3, chaperonin subunit 5, solute carrier family 19a1 and others were identified. Furthermore, 20% of regulated genes were known c-Myc responsive genes (“R”) or their relatives (“rR”). Examples include hexokinase 2, Gpi1, helicase, RNA polymerase 1-3, replication factor C (activator1) 4, Nol5a, Impdh2 and Gart (Table 4). Since the induction of these genes had been repeatedly reported for different tumor cell lines in response to c-Myc activation, they can be considered as likely candidate genes in c-Myc-induced carcinogenesis. It is of considerable importance that the genetic disease model, as described herein, enabled identification of novel c-Myc-responsive genes involved in cell growth and proliferation. For instance, arginase1, ribonucleotide reductase M1 and M2, uridine-cytidine kinase2, topoisomerase (DNA) 2a, meiotic recombination 11a, nucleoplasmin 3, nucleolar proteins and importin4 are novel c-Myc responsive genes whose altered expression is linked to lung adenocarcinomas. Remarkably, several genes which are involved in regulation of transcription through chromatin remodeling were detected. Specifically, up regulated helicase, Smarcc1, histone H1fX, laminB1 as well as the repressed histone1, H2bc and negative regulators of histone acetylation Satb1 and Anp32a were observed. Additionally, in tumors significant increase of the SNRP transcript was identified, which belongs to the dsRNA binding protein family, involved in translation, RNA editing and stability. These findings suggest that chromatin remodeling and posttranscriptional RNA-editing may be additionally mechanisms by which c-Myc augment expression of numerous responsive genes. A further search for c-Myc-recognition sites in promoter regions (2000 bp upstream and 100 bp downstream of the tentative transcription start) of regulated genes was carried out. For this purpose, a specific set of mononucleotide weight matrices with E-box as a core sequence and other computational tools available in the TRANSFAC® professional database (Kel et al., 2003) was used. The results are depicted in FIG. 6 and are also included in Table 4. With the stringent cut-offs applied c-Myc binding sites were identified in promoters of known c-Myc target genes, e.g., Fasn, Shtm1, Srm, Apex1; notably, recognition sites in putative gene targets for c-Myc like Uck2, Hk1, Gapd, Smarcc1, Npm3, Nol5, Ppan, Rnac, Lamr1 and others (Table 4) were identified.

Importantly, no qualitative differences could be determined in the gene expression profiles of papillary adenocarcinomas of various sizes. However, quantitative differences in expression of Tk1, Top2a, Hmgb2, Rrm2, Kpna2, H1fx were observed which were found to be most strongly induced in small-sized adenocarcinomas. This suggests a specific role of these genes at the onset of tumor growth. Other genes, i.e. arginase1, hexokinase2 and anion exchanger Slc4a4 were expressed at higher level in large tumors (more than 2 fold) when compared with small sized tumors pointing to their essential function in malignant growth (Table 4).

It is also of considerable importance that induction of genes in tumors was identified whose expression was absent in non transgenic control lungs (marked by a gray background for the gene title in Table 4); some were consistently increased in all 10 tumors studied (marked by grey rows accordingly in Table 4). Likely, these uniquely expressed genes such as arginase1 and serine hydroxymethyl transferase, are good candidates for novel tumor therapy.

Also, reverse transcription-polymerase chain reaction (RT-PCR) for 12 genes was performed to confirm their regulation in c-Myc induced lung tumors by an alternative method. Notably, the results from both methods agreed well (FIG. 7, Table 5). Additionally, expression of coded protein for Fasn, Shtm1, Arg1 and Hk2 with important functions in cell proliferation and growth was studied. Western blot analysis confirmed the coded proteins (see FIG. 8) to be overexpressed in lung adenocarcinomas.

The solid tumors isolated from the lungs of c-Myc-transgenic mice were identified as papillary lung adenocarcinomas arising from lung epithelial cells, as discussed in part 1 of the study. To follow gene expression changes during lung tumor progression RNA was prepared from tumor sets of small, middle and large size (see Methods) and analyzed using the oligonucleotide microarrays MG u74Av2 to study simultaneously nearly 12500 genes. Statistical analysis of replicates with T-test and stringent comparative ranking was performed to identify regulated genes in tumors as compared with non transgenic healthy lungs (see Methods). The analysis was confined to up regulated genes which were expressed in all tumor samples of at least one set of tumors and down regulated genes which were expressed in all non-transgenic control lungs. The thresholds for significance testing was defined as mean FC>3, p-value in T-test <0,05 and 100% “Increase” call in comparative ranking analysis at least in one set of tumors for up regulated genes and FC ≦−3, p-value in T-test ≦0,05 and 100% “Decrease” for down regulated genes (see Methods). 162 genes with significantly increased expression and 301 genes with significantly decreased expression in c-Myc-expressing lung adenocarcinomas were further analyzed in regard to their biological functions and known c-Myc responsiveness based on publicly available databases and the presence of in silico determined c-Myc binding sites in their promoters. Here the focus was directed on regulated genes involved in extracellular signaling, adhesion, angiogenesis and invasion. In Table 6a, b regulated genes are divided into functional groups. Also, the number of c-Myc-binding sites and known c-Myc responsiveness for each gene analysed is reported. Remarkably, in tumors regulation of many genes was observed implicated in the main extracellular signaling pathways thereby contributing to cell proliferation. Thus, some genes coding for proteins involved in the Ras/Raf/MEK/ERK signaling cascade like the autocrine growth factor amphiregulin, Map2k1 (MEK) were specifically up regulated, and several genes that negatively influence Erk signaling like phosphatases Dusp1, Ptpre and Rassf5, a proapoptic Ras-effector, were found to be repressed. Elevated gene expression of Arhu and Frat2 as well as repressed expression of Tcf7 and Sox, involved in Wnt/beta-catenin signaling was detected. Importantly, in lung adenocarcinomas multiple gene expression changes were found which can lead to excess of₌survival signals and inhibition of antigrowth signaling and, in such a way, contribute to the emerging of a malignant phenotype. These changes included up regulation of Tnf receptor associated factor 4 (Traf 4) and down regulation of the death receptor protein Tnfrsf6 to result in reduction of signaling through cell death receptor. Remarkably, the numerous gene transcript repressed in c-Myc-over expressing lung tumors included many coding for growth-inhibiting functions. Obviously disabling growth inhibitor function contributed to malignant transformation in the lung. Importantly, among repressed genes several genes coding for proteins interfering with survival signaling pathway were detected. These included Igfbp 4, Igfbp5, Igfbp 6 and inhibitor of NFk-beta Nfkbia with reduced expression leads to excessive survival signaling through IGFR and in such a way to suppression of the apoptic capacity of c-Myc.

Furthermore, in lung adenocarcinomas of transgenic mice a considerable decline in expression of genes involved in anti-growth TGFbeta signaling like Bmp6 and Tgfβ1 conveying antigrowth signals, Acvrl1, a member of Tgfβ receptor family, Gadd45b and others was found. Genes whose expression was decreased in lung adenocarcinomas also included inhibitors of proliferation like Gas3, Ndr2, Lox, Meox2, Ptprb and Ptprg as well as transformation repressors like Lats2, scaffolding proteins Akap 12 and caveolin-1.

In c-Myc-over expressing lung tumors repression of genes coding for cell-cell and cell-ECM junctions like laminin alpha 3, integrin alpha 8, cadherin 5, protocadherin alpha 4 and 6, and others was detected. Up regulation of Ect2 and down regulation of Vsnl1 and calpain2 in lung tumors demonstrates a role for c-Myc in regulation of cytoskeleton dynamics of transformed cells.

Increased expression of several proteases like HGF activator, Adam 19, lipocalin, hepsin, thimet oligopeptidase 1 and down regulation of Reck, visinine-like1 and semaphorins may well contribute to invasion and angiogenesis in c-Myc-induced lung tumors.

Some of the differently regulated genes were uniquely expressed in tumors_(expressed in all tumor samples, not detected in healthy non-transgenic control lungs) and coded for extracellular proteins, for example, HGF activator, thimet oligopeptidase and kininogen, which makes them good candidates as tumor markers. Inversely, expression of some other genes detected in all normal lungs was lost in all tumor samples. These included, among others, genes coding for cell adhesion proteins protocadherin alpha 4 and 6, Lama3, procollagen XIIIalpha1, dermatopontin, claudin5 as well as antiproliferative proteins like Bmp6, Ndr2 and Igfbp4, Sema3f, fibulin1, the loss of which in lung epithelial tissues may be essential for the development towards malignancy. These genes are highlighted in the Table 6 a,b.

Quantitative differences in gene expression from small to large size tumors included 2-3 fold changes in expression level of several genes. Among those genes whose expression level was substantially higher in small size tumors were adenosine A2b receptor and kininogen presumably involved in angiogenic signaling. In large size tumors expression levels of proto-oncogene Ros1, protease Hgfac, procollagen type XV and claudin2, were significantly increased, while gene expression of Tgfb1, Gadd45b, Igfbp6, Akap12, Ptpns1 coding for proteins involved in inhibition of growth were significantly decreased when compared with small size tumors that infers an important role for tumor progression.

Among differently regulated genes in lung adenocarcinomas some known c-Myc-responsive genes as Tnfrsf6 and Hoxa5, involved in Tnf-signaling, scaffolding proteins Akap12 and Cav, phosphatase Dusp1, ECM enzyme Lox as well as established c-Myc targets which included both up regulated (for example, mucin1, Icam1) and down regulated genes (for example, Tcf7, Notch4) were found. The remaining regulated genes represent newly identified c-Myc responsive genes and are of great interest for their role in cancer development.

Search of the putative c-Myc-binding sites in the promoter region yielded new putative c-Myc-targets which may play a role in tumor development like Arhu involved in Wnt-signaling, anti-apoptic Traf4, membrane-anchored peptidase Adam19, anti-growth Bmp6 and Rbp1, anti-invasive Reck and previously unsuspected classes of c-Myc-responsive proteins-connexins (Gjb3, Gja1) and semaphorines such as Sema3c.

To confirm the microarray data RT-PCR analysis for 5 up regulated and 6 repressed genes in 11 individual lung tumors and 4 control lungs (FIG. 9) was performed. Gene expression identified by both methods were in good agreement (Table 7). Western blotting (FIG. 10) provided further evidence for regulation at the protein level, most notably for claudin2, Traf4 and Adam19 while proteins Hepsin, Alk1 (Acvrl1) and Igfbp6 were equally expressed in tumors and control lungs.

Hispopatology

By use of the surfactant protein C promoter it was possible to selectively target alveolar epithelium and achieved >50-fold c-Myc expression. This led to an atypical proliferation with malignant cell transformation resulting ultimately in invasive PLACs. The WHO-Classification of human lung tumors (Travis et al., 1999) was used because it fitted well with the observed alterations in c-Myc-induced lung cancer. The resulting hyperchromatic monolayers of bronchiolar alveolar cells were typical for a non-mucinous BAC. The growth of the invasive PLAC was not hindered by the surrounding non-invasive BAC-formations. All invasive tumors of the c-Myc-transgenic model were invariably pure PLAC from the very onset of invasion. Likely, the non-mucinous BAC and pure PLAC observed in the study are derived from the same ancestral tumor progenitor, as suggested by others (Shimosato et al., 1982).

Based on histopathology, the disease model as described herein is highly relevant for the study of human lung malignancies. This is of considerable importance as primary lung adenocarcinoma (LAC) is on the rise. Furthermore, the c-Myc-transgenic disease model mimics subtypes of LAC and, therefore, enables novel insight into the molecular pathology of lung cancer.

c-Myc Activates Cell Cycle

In c-Myc overexpressing lung epithelial tumor cells an altered expression of some key cell cycle regulators was found that can be responsible for the cell cycle reentry as discussed below.

G1/S Promotion

The progression of cell cycle is catalyzed by cyclin-dependent kinases which are activated by cyclins. In c-Myc-induced lung tumors Cdk4 was up regulated, and gene expression of its inhibitor, Cdkn2d (p19), was repressed. Additionally, the expression of cyclin D1 was also increased in tumors. Further, expression of the heterodimer partner of E2Fs, transcription factor Dp1 (Tfdp1), was significantly elevated. These changes are highly suggestive for c-Myc-over expression in the lung tumors to abrogate Rb protein/E2F regulated pathway which controls G1 progression and S-phase initiation and therefore allow inappropriate reinititiation of DNA synthesis.

G2/M Promotion

The final M-phase of cell cycle is triggered by Cdk1 (Cdc2a) associated with mitotic cyclin B. In constitutively c-Myc expressing lung tumor cells, significant up regulation of genes coding for both cyclin B1, cyclin B2 and associated kinase cdc2a (cdk1), the complex of which promotes the transition G2/M, was observed. Besides, over expression of several other genes essential for progression of mitosis like serine/threonine kinases Nek6 and Stk6 (Aurora-A kinase), Cks1 (cdc28 protein kinase), Cks2 (cdc28 protein kinase regulator subunit 2), cdc20, regulators of cytokinesis Prk1 (protein 1), and three kinesin family members coding for molecular motors involved in various kinds of spindle dynamics was detected in lung tumors. Most of these mitotic genes were shown the first time to be activated in response to c-Myc.

Loss of Intracellular Control of Cell Division

Inactivation of p53-mediated apoptosis: C-Myc was found to be a powerful inducer of apoptosis. Moderate elevation of Trp53 gene transcription level in lung tumors of transgenic mice was observed that was expected because the gene is a known direct target for c-Myc. Its expression may be induced by c-Myc directly or indirectly, as a response to signaling imbalance provoked by the oncogene. Importantly, in lung adenocarcinomas deregulation of several genes that interfere with the p53 response was identified. Among these a dramatic decrease of gene expression of epithelially enriched transcription factor Klf-4, an essential mediator of Trp53 in controlling cell cycle progression following DNA damage was detected Repression of intrinsic apoptotic machinery:

In addition to genes linked to the silencing of Trp53 activity, changes in expression of several genes interfering with the cell apoptotic machinery were observed in tumor tissues. One of these was up regulation of Birc5 (survivine), which is a member of the inhibitor of apoptosis protein (IAP) family and interferes directly with the function of caspases (Chiou et al., 2003). Several down regulated genes in tumors coded for proteins interfering with the anti-apoptotic Bcl-2 protein. This is of particular importance because Bcl-2 inhibits almost all types of cell death. One of these repressed genes was Bnip-2, coding for a protein which interacts directly with death-inhibiting Bcl-2 and induced apoptosis via a caspase-dependent mechanism (Zhang et al., 2002). The other repressor calpain-2 is an intracellular protease that was demonstrated to promote decrease of Bcl-2 proteins by cleavage and thereby triggers the intrinsic apoptotic pathway (Gil-Parrado et al., 2002). Proapoptotic effect of Ddit3 (=Gadd153) was linked to down regulation of Bcl-2 and enhanced oxidant injury (McCullough et al., 2001). Furthermore, the tumor suppressor Lats2 was down regulated which was reported to induce apoptosis in lung cancer cells through decrease of the protein level of anti-apoptotic proteins BCL-2 and BCL-x(L) and activation of caspase 9-cascade (Ke et al., 2004). At last, expression of the tumor suppressor Anp32a (=PHAP) was reduced which promotes caspase-9 activation after apoptosome formation (Jiang et al., 2003). Markedly, some of these proteins with regulatory role in apoptosis, have other functions that can also contribute to cell proliferation and motility. For example, Birc5 is implicated not only in anti-apoptosis but also in cytokinesis by stimulating Aurora-B kinase activity (Chen et al., 2003a), Lats2 inhibits cell cycle by controlling different check points (s. above). Anp-32 is involved in repression of transcription as part of the INHAT (inhibitor of histone acetyltransferases) complex (Seo et al., 2002) and calpain-2 plays a role in cell migration through regulation of membrane activity and morphology (Franco et al., 2004).

In summary, the gene expression changes discussed above indicate essential molecular events that allowed c-Myc-overexpressing tumor cells to suppress intracellular control for cell division.

The Presence of c-Myc-Binding Sites in Promoters of Deregulated Gene

C-Myc binding sites were not always identified in genes whose expression was altered in response to c-Myc over expression. Likely, some genes changed their expression as a result of regulation by other transcriptions factors targeted by c-Myc (see Table 2a,b). It was of no surprise that most of the down regulated genes did not contain c-Myc-binding sites in their promoters. Indeed repression of gene transcription by c-Myc is thought to occur independently of binding to the E-box, but is driven by interaction of c-Myc with other transcription factors, notably Miz-1, which recognizes other regulatory sequences (Wanzel et al., 2003). Besides, the repression of several transcription factors by c-Myc, some of them, E-boxes containing, may be one of the reasons for the remarkable large number of down regulated genes in c-Myc-induced lung tumors.

Unexpectedly, a small number of genes known to bind c-Myc directly, e.g. cyclin B1, Cdc2a, Cks2, did not contain c-Myc recognition sites in the bioinformatic analyses. This requires further consideration. First, the selected promoter region was restricted to 2000 bp upstream and 100 bp downstream of the putative transcription start site. It is possible that some c-Myc binding sites are positioned outside this region. Second, the matrices similarity cut-offs were set stringently so that false positive matches would be less than 1 per 10,000 bp. On the other side, such cut-off enables recognition of only 10-30% of putative sites. Finally, for human E-box containing genes a distribution into two groups with low and high affinity binding for c-Myc has been reported (Fernandez et al., 2003). Remarkably, overexpression of c-Myc enhanced its association with low affinity E-boxes and at very high level to non targeted chromatin. Possible, c-Myc overexpression of >50-fold, as achieved in the study, resulted in perturbation of transcriptional control and extended activity of c-Myc beyond E-box containing promoters. The consequence may be up regulation of such genes as Birc5, Ect2 and others found to be uniquely expressed in lung tumors.

Orthologs Regulated in Human Malignancies.

A number of genes regulated in c-Myc-induced papillary lung adenocarcinomas that contribute to cell cycle progression, growth and block of apoptosis, i.e. Ccnd1, Stk6, Klf-4, Gas1, Hey1 and Stat-1 were similarly regulated in various human malignancies (He et al., 2004; Anand et al., 2003; Chen et al., 2003a). Apart from remarkable agreement in histological phenotypes the disease model mimic closely genetic events in human lung cancer. Therefore, its significance in the study of lung tumor development is emphasized. Specifically, altered expression of some genes in lung tumors of the mouse model, were identified in human NSCLC as well. This included overexpression of CDK4, CDC2A, TFDP1, CCNB1, CDC20 (Singhal et al., 2003), and contributed to promotion of cell cycle. In addition, overexpression of anti-apoptotic BIRC5 (Escuin and Rosell, 1999) and heat shock protein HSP70 (Volm et al., 1995) as well as repression of the tumor suppressor LATS2 (Li et al., 2003) were also found in human lung cancer. Up regulation of Cdk4 and down regulation of pro-apoptotic Bnip2 were likewise detected in chemically induced murine lung tumors (Bonner et al., 2004). These changes in gene expression point to similarities in the development of lung adenocarcinoma in mouse and human malignancies and demonstrate the relevance of the c-Myc-induced lung tumor model for investigating human NSCLCs.

To the best of our knowledge, some genes identified in the study were not previously associated with human lung carcinogenesis like the newly identified c-Myc responsive genes Hey-1, Anp32a, Bnip-2, calpain-2, amongst others. Their altered expression may lead to inhibition of apoptosis and these genes represent interesting candidates for further validation as therapeutic targets.

Central Role of c-Myc in Activation of Cell Metabolism

Previously, changes in gene expression induced by targeted c-Myc-overexpression in lung epithelium were reported to result in an activation of genes coding for cell cycle promotion and inhibition of cell cycle arrest and death. Here the oncogenomics associated with different metabolic pathways are presented to enable rapid growth and proliferation of tumor cells. Notably, induction of genes coding for DNA and RNA metabolism, protein synthesis, ribosomal biogenesis, glycolysis and transport in c-Myc induced lung tumors was observed (see Table 4). The ability of c-Myc to control transcription of genes involved in both cell cycle progression and growth is in agreement with other studies (Lutz et al., 2002). A significant number (53,7%) of identified genes are known to be c-Myc responsive in other cell types (FIG. 6). This supported the notion that c-Myc is a key activator for cell metabolism and that the set of regulated genes is, at least in part, common in various cell types. Moreover, the presence of c-Myc recognition sites in more than one half of regulated genes (Nonetheless Nonetheless.1) argues for the ability of c-Myc to directly activate the transcription of genes directly involved in cell growth.

Metabolic Genes Activated by c-Myc Contributes to Regulation of Cell Proliferation

Recent data provided evidence that several c-Myc target genes which were found to be upregulated in lung adenocarcinoma code for metabolic enzymes and may contribute otherwise to cell proliferation. For example, thymidilate synthase which maintains the dTMP pool critical for DNA replication functions also as an RNA binding protein. It forms a complex with a number of cellular mRNAs, including the RNA corresponding p53 tumor suppressor gene, to cause translational repression (Liu et al., 2002), and, in such a way, may regulate cell cycle and apoptosis. Furthermore, fatty acid synthase (Fasn) is a key metabolic enzyme catalyzing the synthesis of long-chain saturated fatty acids and plays a central role in the production of surfactant in lung. On the other hand overexpression of Fasn has been linked to dysregulation of membrane composition and modulation of lipid rafts functioning in tumor cells. Lipid rafts are membrane microdomains involved in signal transduction, intracellular trafficing and cell migration (Swinnen et al., 2003). The Fasn is expressed at high level in numerous human malignancies (Evert et al., 2005), and it was found that the ability of flavonoids to induce apoptosis in cancer cells is strongly associated with their properties to inhibit Fasn (Brusselmans et al., 2005). In addition, nucleophosmin (Npm1) is a nucleolar phosphoprotein which functions as a molecular chaperone and plays an important role in ribosomal protein assembly and transport through prevention of proteins from aggregating in the nucleolus. Besides, it inhibits apoptosis by suppression of the protein kinase PKR. This kinase effects apoptosis in normal cells in response to various extra and intracellular cues (Pang et al., 2003). Up regulation by c-Myc of spermidine synthase (Srm) in lung tumor cells could lead to elevated intracellular level of polyamines which are substantial for cell proliferation. Polyamines are not only of critical importance for protein biosynthesis, membrane stability, chromatin structure or modulation of ion channels but they have been shown in breast cancer cells to regulate the interaction of transcription factors such as NFkB with their responsive elements, thereby leading to an up regulation of genes involved in proliferation (Shah et al., 2001). In the case of Fas, Npm1 and Srm multiple (2,3 and 5, respectively) c-Myc-binding sites were found in the promoter region (Table 5). This suggests direct up regulation of these genes by c-Myc in lung adenocarcinomas. Unlike normal cells many tumors rely entirely on glycolysis for ATP production even in a not hypoxic environment, known as the Warburg effect (Garber, 2004). Though being less efficient, glycolysis provides ATP quickly which is needed for the many metabolic processes associated with rapid tumor growth. Note, several glycolytic enzymes were over expressed in lung adenocarcinoma and contained c-Myc-recognition sites in their promoters or were previously shown to bind c-Myc directly (see Table 1). This clearly demonstrates a direct role of c-Myc in the activation of glycolysis (Table 4). It is highly interesting that glycolytic enzymes, similarly to other metabolic enzymes discussed above, have been shown to have additional functions in cell proliferation. For example, activity of hexokinase 2 associated with the outer membrane of mitochondria, has been implicated in protection from apoptosis (Garber, 2004; Majewski et al., 2004). Gpi1/AMP (glucose phosphate isomerase 1/autocrine motility factor), a ubiquitous cytosolic enzyme that plays a key role in glycolysis, is a multifunctional protein that acts in the extracellular milieu as a potent mitogen/cytokine. Indeed, its over expression contributes to motility, invasion and metastasis (Garber, 2004; Tsutsumi et al., 2004) and was correlated with aggressive tumor growth and poor prognosis in human pulmonary adenocarcinomas (Garber, 2004; Takanami et al., 1998). These examples demonstrate how expression of metabolic genes induced by c-Myc contribute to promotion of cell proliferation in lung tumorigenesis.

Remarkably, 9 genes coding for enzymes involved in purine and pyrimidine nucleotide synthesis pathways (Tk1, Tyms, Shtm1, Srm, Impdh2, Gart, Uck2, Rrm2, Rrm1) were identified as well as folate transporter Slc19a1 which were found to be over expressed in lung adenocarcinomas and are essential for rapid cell proliferation. Indeed, inhibition of thymidilate syntase by 5-fluorouracil is an established therapy against certain tumors. Notably, Shmt1, both mRNA (Table 4) and protein (FIG. 8), was expressed in all c-Myc-induced lung adenocarcinomas but not in healthy non-transgenic lung. Its role was examined in human breast cancer cells and it was shown that cSHTM1 is a metabolic switch that, when activated, gives dTMP synthesis higher metabolic priority than competing synthesis of SAM (S-adenosylmethionine), a cofactor that methylates DNA, RNA, proteins and many metabolites (Herbig et al., 2002). Furthermore, among other genes which were uniquely expressed in lung adenocarcinomas five genes coded for enzymes involved in DNA metabolism—e.g. thymidine kinase 1, topoisomerase (DNA) II alpha, helicase, ribonucleotide reductase M1 and meiotic recombination 11 homolog A. These genes were expressed at the highest level in small tumors. Again, this points to their essential role in emerging tumors. Up regulation of Apex and Xrcc5, involved in DNA repair, indicate that c-Myc couples DNA replication to the maintenance of DNA integrity.

Altered expression of several genes coding for proteins important in chromatin remodeling.

The increased expression of linker histone H1fx and repression of core histone H2b1 could change the structure of nucleosome and access to nucleosomal DNA for gene expression, DNA replication and repair. Besides, increased expression of genes coding for helicase and Smarcc1 was found. Both have c-Myc-binding sites in their promoters and are involved in all processes including DNA strand separation like gene transcription, DNA replication, recombination and repair. Satb1 and Anp32a, which are found to be repressed, are involved in repression of gene transcription through interfering with histone acetylation (Seo et al., 2002). Altered expression of such genes could lead to remodeling of large chromatin domains and initiate undue-expression of numerous genes in c-Myc-overexpressing cells. In addition, elevated level of the SNRP transcript, the gene coding for a protein involved in RNA editing (the evolutionary conserved mechanism for regulation of gene expression at the posttranscriptional level that plays an essential role in development) may prove to be of great importance for lung carcinogenesis. These genes were identified as new c-Myc responsive genes and first demonstrate their regulation in cancer (except for helicase which was linked to cancer previously).

Orthologues Regulated in Human Malignancies.

Several genes regulated in c-Myc-induced lung adenocarcinomas contribute to cell growth e.g. Hk2, Fasn, Uck2, Impdh2, Tk1 and were shown previously to be similarly regulated in human malignancies (Shen et al., 1998; Swinnen et al., 2003; Goel et al., 2003; Natsumeda et al., 1990). Strikingly, altered expression of some of these orthologues were specifically identified in human NSCLC. This included induction of RRM2 and TOP2A transcript expression with defined roles in DNA synthesis and transcription (Wikman et al., 2002), as well as overexpression of ARG1 which fosters polyamine synthesis (Suer et al., 1999). High activity of TYMS activity and GPI1 expression was associated with poor prognosis of patients with NSCLC (Nakagawa et al., 2004; Takanami et al., 1998) as was exaggerated activity of LDH with tumor stage (Rotenberg et al., 1988). Clearly, this demonstrates the relevance of the findings obtained in transgenic mice for human NSCLCs, as described herein. To the best of our knowledge, certain genes identified in the study were not previously associated with human lung carcinogenesis and these included the c-Myc targets Srm, Shmt1, Npm1, Slc19a1 as well as the newly identified c-Myc responsive genes Satb1 amongst others. Their specific regulation represents new and important therapeutic targets worthy for further exploration to combat this highly malignant disease.

In the effort to better understand the transforming capacity of c-Myc in lung adenocarcinomas firstly gene networks directly involved in cell cycle progression and cell growth as well as regulation of cell cycle arrest and programmed cell death were examined. As intrinsic programs to regulate cell proliferation, growth and cell death are also governed by extracellular signaling, regulation of genes coding for proteins involved in the main pro- and anti-growth extracellular signaling pathways was studied.

In the following, the consequences of c-myc overexpression on extracellular signaling, angiogenesis and invasion are discussed.

Incapacitating of Extrinsic Pathways of Growth-Inhibitory Signaling

Decrease of Cell Death Receptor Signaling

In lung tumors gene expression changes were identified that could have a negative impact on the death receptor signaling, for example, decrease in expression of the death receptor protein Tnfrsf6 (FAS) and up regulation of uniquely expressed gene transcripts in tumors, such as Traf 4 which codes for a putative signal-transducing protein and was shown to inhibit Fas-induced apoptosis in transformed embryonic kidney cells (Fleckenstein et al., 2003). Both Tnfrsf6 and Traf 4 possess c-Myc-binding site in their promoters, and presumably are directly regulated by c-Myc. Besides, in lung tumors decreased expression of proapoptic transcription factor Hoxa5 was found which was shown to sensitize cells more than 100-fold to TNF-alpha-induced death mediated by caspase 2 and 8 (Chen et al., 2004). Expression of this transcription factor led to apoptotic death in breast tumor cells and was lost in the majority of breast tumors (Raman et al., 2000). The expression of these genes could make lung epithelial cells resistant to apoptosis induced by cytokines like FasL and TNFalpha.

Evading the Tgf-Beta Growth-Suppression Signaling

Gene expression of several components of the Tgf-beta (TGFb) signaling pathway was significantly repressed in c-Myc-induced lung adenocarcinomas. Members of the TGFb superfamily, including TGFb and BMPs are important extracellular signaling proteins which inhibit proliferation of several cell types, either by blocking cell cycle progression through G1 or by stimulating apoptosis. Down regulated genes in lung tumors of transgenic mice included genes coding for type I receptor for TGFb superfamily of ligands Acvrl1 and a Tgfb coreceptor endoglin as well as the ligand molecules Tgfb1 and Bmp6. Notable, gene expression of Bmp6, a new putative c-Myc target, was lost in all lung tumor tissues of transgenic mice. Additionally, expression of dermatopontin was completely abrogated in lung tumors. This gene codes for a component of extracellular matrix that increases the cellular response to TGF-beta (Okamoto et al., 1999). Besides, significant decrease in expression of transcription factor Zfhx1a that is crucial regulator of TGFb/BMP signaling through synergizing with SMAD to induce growth arrest (Postigo et al., 2003) was detected. Additionally, an immediate early response gene for TGFb inhibitory growth factors Gadd45b, which functions as a positive effector of Tgfb-induced apoptosis (Yoo et al., 2003), was found to be repressed in tumors of c-Myc transgenic mice. Gadd45b is a likely c-Myc target gene based on the c-Myc recognition site which was identified in its promoter. A strong repression of Gadd45 was also observed in RAT-1 overexpressing c-Myc cells (Amundson et al., 1998) and therefore agrees well with the finding as described herein. The inhibited expression of discussed genes could likely disrupt growth-inhibitory signaling by Tgfb family proteins in lung tumor tissues.

Promotion of Cell Survival Through IGFR-Signaling Pathway

Apoptosis can be antagonized by exogenous survival factors like insulin-like growth factors (IGF) I/II which bind to cell-surface receptors and activate PI3 kinase-Akt/PKB signaling pathway that keeps the cell death program suppressed. Gene expression changes in c-Myc induced lung adenocarcinomas indicated significant activation of the cell survival signaling pathway. The prominent feature in lung adenocarcinomas was the significant decrease of gene expression levels of three Igf-binding proteins—Igfbp 5, 6, 4 which were expressed at high level in control lungs. These IGFBPS bind to IGFs and regulate its biological availability and function. Besides, they exert also IGF-independent growth-inhibitory effects (Clemmons, 1998). Thus, decline of the level of the IGFBPs could provide for a local excess of available IGFs in tumor tissue and thereby contribute to antiapoptotic signaling through IGFR. It is of considerable importance that amongst several growth factors tested only IGFs proved to be survival factors for c-Myc-over expressing fibroblasts (Harrington et al., 1994). Furthermore, repression of additional genes involved in negative regulation of IGF1R signaling pathway was found. These were Socs2 coding for a protein which interacts with IGF1R and suppresses IGF1-induced growth (Dey et al., 1998) and Nfkbia, which inhibited NFkB (a transcription factor activated by PI3K-Akt signaling) by trapping it in the cytoplasm. Activation of IGFR-mediated survival signaling through inhibition of expression of several negative regulators of the pathway could antagonize c-Myc-mediated apoptosis and allow proliferation and growth of c-Myc-overexpressing lung epithelial cells without concomitant apoptosis.

Activation of Growth Factor Signaling Through Tyrosine Kinase Receptors and Related Genes

Many of the regulated genes identified in c-Myc-induced lung adenocarcinomas coded for molecules involved in the main cell signaling pathways with a positive effect on cell division and growth. A key role in extracellular stimulation of cell growth and proliferation belongs to the signaling through EGFR (epidermal growth factor receptor). One of the up regulated genes in lung adenocarcinomas of c-Myc transgenic mice coded for amphiregulin (AR), an autocrine growth factor of the EGF family. AR was shown to stimulate proliferation of human lung epithelial cells through binding to EGFR (Lemjabbar et al., 2003) as well as survival of the human NSCLC cells through activation of IGF1R-dependent pathway (Hurbin et al., 2002). Besides, AR was demonstrated to be an essential mediator of G-protein-coupled receptor (GPCR)-induced transactivation of EGFR signal and down stream cell response in squamous cell carcinoma. In turn, the secretion of AR could be induced through an activation of the EGFR-signaling pathway in bronchiolar epithelial cells (Blanchet et al., 2004) and IGF1R in NSCLC (Hurbin et al., 2002). These findings suggest that AR growth factor may play an important role in lung cancer development and, like in human cells, increased AR expression in c-Myc-induced lung tumors may resulted in stimulation of autocrine growth and suppression of apoptosis.

One further component of the EGFR/Ras/Raf/Mek/Erk-signaling cascade found to be up regulated in lung tumors was Map2k1 (Mek) while Rassf5, the pro-apoptic Ras effector, was down regulated, therefore evidencing the suppression of Ras-mediated apoptotic signaling. Notable, Rassf5 was not expressed in 80% of human lung adenocarcinomas and was specifically repressed in lung epithelial tumor cells (Vos et al., 2003). Additionally, among down regulated genes in lung adenocarcinomas several phosphatases like Dusp1, Ptpre, Ptpns1, Ptprb and Ptprg were identified. Likely, specific dephosphorylation of signaling proteins such as MAPKs negatively regulate receptor tyrosine kinase-coupled signaling processes. Notably, the phosphatases Ptprb and Ptprg excert growth inhibitory effect (Gaits et al., 1995); (Liu et al., 2004), and expression of Ptprb was dramatically decreased expression of Ptprb in human lung adenocarcinomas (Gaits et al., 1994).

Among uniquely expressed genes in c-Myc-induced tumors the proto-oncogene Ros1 coding for a transmembrane tyrosine kinase which may function as a growth factor receptor (Keilhack et al., 2001 was identified).

Another gene uniquely expressed in tumors coded for the HGF activator, a secreted protease that converts precursor of HGF in its active form which functions as a potent mitogenic factor for epithelial cells and was found to induce angiogenesis (Sengupta et al., 2003). Up regulation of HGFac was also observed in human lung adenocarcinomas (McDoniels-Silvers et al., 2002). The unique expression of these genes which in tumorous lungs may be essential for promotion of lung carcinogenesis. Besides, up regulation of Adam19, a member of a disintegrin and metalloprotease family that is involved in signal transduction through processing of transmembrane growth factor precursors (Fischer et al., 2003) may foster activation of EGFR-signaling in lung tumors of transgenic mice. Presence of c-Myc-binding sites in the promoters of Adam19, Dusp 1 and Map2k1 likely indicates their direct regulation by c-Myc.

Activation of Wnt/Beta-Catenin Signaling

The study of differently expressed genes in lung tumors of transgenic mice provide evidence for Wnt signaling to be affected in c-Myc-induced lung adenocarcinomas. In tumor tissues enhanced expression of Arhu, a member of the Rho family that mediates in part the effects of Wnt1 signaling in the regulation of cell morphology, cytoskeleton organization and cell proliferation (Tao et al., 2001) was found. Also, unique expression of Frat2, a positive regulator of Wnt signaling and stabilizer of beta-catenin (Saitoh and Katoh, 2001) was observed. In contrast, repressed expression of the transcription factors Tcf7 and Sox7, which are negative regulators of Wnt/beta-catenin-induced gene transcription, was observed. Down regulation of these genes was shown to be associated with malignant transformations (Roose et al., 1999). Modulation of Wnt signaling in c-Myc transgenic mice contributed to promotion of cell proliferation, cytoskeleton dynamic and motility of tumor cells. It is of considerable importance that c-Myc-binding sites were found in promoters of Arhu and Tcf7, the latter was shown to be a c-Myc target gene, therefore linking c-Myc directly to the activation of Wnt-signaling pathway.

Regulation of Cytoskeleton Contributing to Transformation, Increased Motility and Invasiveness

The ability of malignant cells to migrate and invade surrounding tissues is associated with changed motility based on modifications of the cytoskeleton. A central role in rearrangement of the actin cytoskeleton in response to diverse external signals is ascribed to members of the Rho protein family. Many genes involved in regulation of cytoskeleton and transformation were found to be differentially expressed. These included the strongly up regulated proto-oncogene Ect2, a growth-regulatory molecule, which, in addition to its ability to regulate cell cycle, activates as a guanine nucleotide exchange factor the Rho family of Ras-related GTPases to bring about remodelling of actin cytoskeleton and to contribute to cell transformation (Saito et al., 2004); (Westwick et al., 1998).

In lung tumors the loss of expression of invasion suppressor Vsnl1, which was shown to decrease RhoA activity and MMP-9 level through cAMP-mediated signaling (Mahloogi et al., 2003), may enhance the malignant phenotype. Down regulation of calpain-2, an intracellular protease required for proteolysis of the cytoskeleton and focal adhesion proteins was shown to restrict membrane protrusions and lamellipodia dynamics (Franco et al., 2004), to further strengthen promigratoric capacity of lung tumor cells.

Changes in Cell Junctions and Adhesion: Increased Motility and Invasiveness

Cell-cell and cell-extracellular matrix (ECM) junctions do not only bind cells mechanically through adhesion but also transmit signals to the cell interior thereby playing an important role in regulation of cell survival, proliferation and migration.

For example, up regulation of the c-Myc target gene mucin1, a transmembrane glycoprotein defined as a tumor antigen (VanLith et al., 2002) that has been implicated in activation of Wnt/beta-catenin-signaling (Huang et al., 2003) was observed in lung tumors of transgenic mice. Over expression of mucin1 was shown to be involved in invasive tumorigenesis in the breast (Schroeder et al., 2003) and colon (Baldus et al., 2004). The other c-Myc target gene Icam1 (intercellular adhesion molecule1) which is the only known direct ligand of the mucin extracellular domain was also over expressed in c-Myc-induced lung tumors. It was shown that mucin1, on the contact with Icam1 produced calcium-based signals that might affect the cytoskeleton and increased cell motility (Rahn et al., 2004). In contrast, strong decrease in expression of genes coding for cell-cell junction proteins like cadherin-5, protocadherins alpha 4 and 6 may contribute to cell proliferation and migration in lung tumor. Additionally, in lung adenocarcinomas of transgenic mice repression of several genes coding for proteins implicated in cell-ECM interactions was observed. These included fibulin-1, an extracellular matrix (ECM) protein which affect the Erk-mediated signaling transduction cascades to inhibit motility and invasiveness of cancer cells (Twal et al., 2001), and Lama3 (laminin 5), a ECM glycoprotein shown to be repressed in various human cancers (Sathyanarayana et al., 2003). The decreased expression of matrix receptor alpha8integrin which negatively affects cell migration and proliferation (Bieritz et al., 2003) could therefore contribute to advanced malignant phenotype of lung tumors.

Tumor specific regulation of genes involved in cell adhesion may impact control of cell growth through changes in cell-cell and cell-matrix attachment to foster increased cell proliferation and motility in lung tumor tissue.

Degradation of ECM

Based on the histopathology of PLACs an invasive nature of the lung tumor was evidenced. Note, proteolysis of matrix proteins is essential for cell migration. Matrix components are degraded by extracellular proteolytic enzymes, most of which are matrix metalloproteinases while others are serine proteases. In lung tumors up regulation of gelatinase-associated protein lipocalin 2, a regulator of activity of matrix metalloproteinase (MMP) was found, which was suggested to be involved in invasion of tumor cells (Liu et al., 2003; Huang et al., 2003) Additionally, repressed expression of the metastasis suppressor Reck was observed (Liu et al., 2003) which suppresses invasiveness of carcinoma cells through inhibition of the release of MMP. Likewise, upregulation of the metalloproteinase Adam 19 impacts EGFR signaling as discussed above. Furthermore, the known c-Myc responsive gene Thop1 was significantly overexpressed in tumor tissues of transgenic mice. This gene codes for a member of the metalloendopeptidase family but was not as yet linked to cancer. Among serine proteases over expressed in c-Myc-induced lung tumors the cell surface protease hepsin was found over expressed. This protein was demonstrated to cause disorganization of basement membrane and promote progression of metastasis in a mouse prostate cancer model (Klezovitch et al., 2004). The other serine protease, i.e. HGF activator, a serine protease specific for activation of HGF (Tjin et al., 2004) with key roles in invasion and metastasis of cancer (Hurle et al., 2005), was upregulated as well. At last, gene transcription of extracellular enzyme lysyl oxidase (Lox) indicated an inhibition of cross-linking of collagen and elastin in ECM that makes the matrix fragile and prone to tear. Mutations or reduction of the LOX transcript level in human cancer suggests this protein may excert tumor suppressive activity (Rost et al., 2003).

Gap Junctions

Over expression of connexins 43 and 31 in lung tumors revealed an unsuspected role for c-Myc in cell-cell communication.

Note, there connexins contain c-Myc binding sites in their promoters as evidenced by in silico annotations. These proteins form intercellular channels, which allow the exchange of small molecules (such as inorganic ions, sugars, nucleotides, intracellular mediators like cyclic AMP and IP3) among coupled cells. Connexin43 is essential for survival, and its expression was altered in malignant tissues and in the presence of carcinogenic factors. Besides, it was reported that connexins can be induced through Ras-signaling (Carystinos et al., 2003). The consequences of perturbed connexin 31 expression on cell proliferation (Kibschull et al., 2004) and cell death (Di et al., 2002) were discussed elsewhere.

Tight Junctions

Tight junctions seal epithelial cells together and function as selective permeability barriers. They are formed exclusively by integral membrane proteins of the claudins family. Their role in cancer is not well understood. High level of claudin-5 gene expression was observed in control lung which was progressively suppressed and eventually absent in large tumors. As a consequence loosening of intercellular adhesion and increased motility can be expected. Inversely, gene expression of claudin 2, absent in normal lungs, was strongly induced in tumors, probably, due to the activation of Wnt signaling (Mankertz et al., 2004). Claudin 2 and some other claudin family members were shown to be involved in activation of proMMP through recruitment of enzymes on the cell surface (Miyamori et al., 2001) and so may contribute to invasion.

Proangiogenic Signaling

Some of the differently expressed genes in lung adenocarcinomas of transgenic mice can be linked to angiogenesis. Indeed, a remarkable up regulation of kininogen, a haemostatic factor that exerts its angiogenic effect (Colman et al., 2003) through signaling of its cleaved product form bradykinin through the B (2) receptor, was observed (Yang et al., 2003). Increased transcription level of G-protein-coupled receptor Adora2b detected in tumor tissues might additionally contribute to a stimulation of angiogenesis because it was shown to mediate secretion of angiogenic factors like VEGF and IL-8 (Feoktistov et al., 2003). Furthermore, in all lung tumors the loss of expression of Sema3f, a secreted signaling molecule from the immunoglobulin family with antagonistic action towards Vegf due to competitive binding to neuropilin1/2 receptors (Nasarre et al., 2003), was observed. Sema3f was demonstrated to inhibit angiogenesis (Kessler et al., 2004), metastasis (Bielenberg et al., 2004) and motility of primary tumor cells (Nasarre et al., 2003). Decreased expression of Sema3f correlated with advanced stage and more aggressive type of human NSCLC (Roche and Drabkin, 2001) that indicates its important role in lung carcinogenesis. At last, up regulation of Hgfac in c-Myc-induced tumors could result in activation of HGF, whose multiple functions include also a potent angiogenic capacity.

From the study the inferred link of c-Myc to angiogenesis is in agreement with an observation that—c-Myc had a potent angiogenic capacity. This was shown in skin (Pelengaris et al., 1999), pancreatic β-cells (Pelengaris et al., 2002) and lymphoma (Brandvold et al., 2000). The reversible activation of conditional alleles of c-Myc in these studies induced hyperproliferation, dedifferentiation and angiogenesis in a reversible manner, suggesting that these complex events may be directly affected by c-Myc.

Deregulation of Signaling Pathways Through Scaffold Proteins

Gene expression changes discussed above link c-Myc to the regulation of the main intra- and extracellular signaling pathways. To this question, in lung adenocarcinomas of c-Myc-transgenic mice significantly decreased expression of several scaffolding proteins involved in spatiotemporal regulation of signaling pathways was found. They included two known c-Myc responsive genes caveolin1 and Akap12 (gravin), both proteins being frequently absent in a number of carcinomas (Williams et al., 2003), including lung cancer (Wikman et al., 2002). Caveolin-1 is the principal structural component of caveolae—the sites of membrane responsible for concentrating an array of signaling molecules including growth factor receptors and G-proteins as well as calcium channels and pumps, and thought to be a major regulator of signaling complexes in caveolae. The loss of caveolin-1 was demonstrated to confer a significant growth advantage to mammary epithelial cells in vitro and in vivo (Williams et al., 2004). Decreased expression of the c-Myc responsive genes caveolin-1 together with overexpression of FAS, points to an important role of derangement of the membrane rafts in the malignant transformation induced by c-Myc. The transformation suppressor potential of Akap 12 seems to be based on its ability to reorganize actin-based cytoskeleton architecture and control G1 phase signaling proteins (Gelman, 2002). Scaffold proteins guide the interactions between successive components of signaling complexes to relay the signal with precision, speed and efficiency and to avoid an unwanted cross-talk between signaling pathways therefore providing for a specificity of cell response to many different signals. The loss of such spatiotemporal regulators of signaling processes in response to c-Myc could likely resulted in an amplification and spreading of proliferative signals in lung epithelial cells thereby accelerating malignant transformation.

CONCLUSIONS

-   -   1. As reported in Part 3 and in Part 1 and 2 of the c-Myc         oncogenomic study according to the invention, inappropriate         expression of a variety of genes involved in the several         pathways critical for the development of lung tumor was         identified: cell cycle, cell growth, cell death, adhesion,         cytoskeleton organization, invasive and angiogenic capacity (see         FIG. 11 for a summary).

2. Some genes identified in this study were known to be similarly changed in human malignancies and specifically in lung tumors which makes the SPC/c-Myc-transgenic mouse model useful for investigating human lung cancer.

3. Many other genes differently expressed in lung tumors are novel targets for mechanism based therapies and serve as useful tumor markers.

4. New c-Myc-responsive genes and putative c-Myc-targets identified in this study contributed to the collective examination of distinct c-Myc transcriptomes that are partly cell type- and species-specific and therefore represent one more important result of this study.

c-Myc is a transcription factor and frequently overexpressed in diverse malignancies. It's targeted overexpression (>50-fold) in mouse alveolar epithelium induced lung cancer. To identify new myc target genes and to explore the molecular basis of c-Myc transforming capacity were investigated genome wide regulation of genes in histologically well defined tumors. Essentially, expression of 463 genes differed significantly when compared with healthy non-transgenic lung. Then promoters of regulated genes for c-Myc recognition sites to link c-Myc binding to gene expression were annotated and interrogated. Notably, many of the genes could be traced back to regulators of cell cycle and included c-Myc targets, e.g. Cdk4, Ccnb1, Cks2 in addition to c-Myc responsive genes, e.g. Tfdp1, Cdc2a, Stk6, Nek 6, Prc1, Ect2. Furthermore, Several genes whose regulation in tumors interfere with the p53- and Bcl2-mediated cell cycle arrest and cell death program were identified, notably Birc5, KLf4, Stat1, Ddit3, Lats2 therefore providing a selective advantage for tumor growth. There was considerable agreement among the regulation of c-Myc target genes in mouse and human lung malignancies. Notably, targeting new lung cancer genes i.e. Prc1, Klt4, Ect2, Hey-1, Gas-1, Bnip-2, calpain-2 and Anp32a may enable mechanism based therapies.

The c-Myc transcription factor regulates a wide set of genes in response to mitogenic stimuli. Its targeted overexpression in alveolar epithelial cells led to malignant transformation and development of papillary lung adenocarcinomas (PLAC) in mice. In the first part of the study cell cycle and apoptosis networks were investigated in papillary adenocarcinomas. Now regulation of genes coding for DNA and RNA metabolism and processing, ribosomal biogenesis and protein biosynthesis, nuclear and cellular transport, glycolysis, lipid metabolism and chromatin remodeling is reported. The c-Myc target genes Tyms, Tk, Srm, Fasn and the c-Myc-responsive genes Hells, Rpo1-3, Gpi1, Impdh were found to be significantly induced. Their regulation in PLAC could contribute to an accelerated cell proliferation and growth. Notably, increased expression of some orthologues in human lung carcinomas was observed as well. In addition, bioinformatics to search for c-Myc-binding sites in promoters of regulated genes were applied thereby identifying new c-Myc targets; these included, amongst others, Uck2, Smarcc1, Npm3, Nol5, Lamr1, next to the well established c-Myc targets Srm, Shmt1, Npm1, Slc19a1. These newly identified lung cancer genes are interesting targets for the development of novel antineoplastic therapies.

To better understand the molecular basis of c-Myc transforming capacity genome-wide expression analysis of lung adenocarcinomas in a transgenic mouse model was performed. In part 1 and 2 of the study regulatory gene networks associated with cell cycle and apoptosis as well as tumor growth and metabolism was reported.

Here it was focussed on regulated genes involved in extracellular signaling pathways, e.g. activation of EGFR-mediated mitogenic, IGFR-mediated survival and Wnt-signaling as well as disruption of Fas-mediated death and anti-mitogenic Tgf-β-signaling. Additionally, altered gene transcription associated with cell adhesion, cell-cell junctions, regulation of cytoskeleton, proangiogenic signaling and invasion provided a molecular rationale for c-Myc invasive and angiogenic capacities. An identification of novel c-Myc target genes associated with lung tumors (e.g., Rbp1, Reck) along with known c-Myc-responsive or c-Myc-target genes (e.g., Akap12, Cav1, Hoxa5, Lox, Muc1) and genes linked to lung cancer (e.g., Rassf5, Hgfac, Sema3f, Ptprb), provide highly interesting targets for mechanism based therapies.

Materials and Methods

Maintenance of the Transgenic Mouse Line

SPC/myc transgenic mice (Ehrhardt et al., 2001) were maintained as hemizygotes in the CD2F1-(DBA/2xBalb/C) background and identified by PCR of DNA extracted from tail biopsies (Hogan et al., 1994) (FIGS. 5 a, b). PCR was carried out using Platinum PCR SuperMix (InVitrogen) with the primer pair for the c-Myc-transgene: 5′-CAGGGCCAAGGGCCCTTGGGGGCTCTCACAG (SEQ ID NO 1), 3′-GGACAGGGGCGGGGTGGGAAGCAGCTCG (SEQ ID NO 2):

Sample Collection and Preparation

Mice were anasthesized by an overdose of CO₂ at the age of 9-13 months. The tumors were inspected macroscopically, separated from the surrounding lung tissue of SPC/myc transgenic mice and frozen immediately in liquid nitrogen. The tumors were divided into groups according to size 1 mm, 5 mm and >10 mm. Small tumors were pooled in three groups of 3, 3 and 4 separate tumors because of the low yield of RNA. Normal lung from four non transgenic mice of about the same age were used as a control.

Histology

For histopathology tissue specimen of 5 transgenic animals and 2 non-transgenic controls at the age of 9 to 13 months were investigated. The specimens were rinsed in PBS and fixed in 4% PBS-buffered formaldehyde, and processed for paraffin embedding as usual. 5 μm thick serial sections were stained with hematoxylin and eosin (H and E), hematoxylin only (H) and PAS for light microscopic evaluation.

Isolation of RNA, Production of c-RNA, Array Hybridization and Scanning.

The cRNA-samples were prepared following the Affymetrix Gene Chip® Expression Analysis Technical Manual (Santa Clara, Calif.). 10 μg of biotinylated fragmented cRNA was hybridized onto the Murine Genome U74Av2 Array (MG-U74Av2)—a GeneChip® expression oligonucleotide probe array from Affymetrix which contains 12 488 probe sets, that represents annotated sequences in the Mouse UniGene database as well as EST clones. The procedures for isolation of RNA, production of c-RNA, array hybridization and scanning were done according to Affymetrix's manual and as described (Borlak et al., 2005). Each hybridization image was scaled to all probes set intensity of 250 for comparison between chips.

Gene Expression Data Analysis

The data analysis was performed basically as described earlier (Borlak et al., 2005). For single arrays a statistical expression algorithm within the Affymetrix® Microarray Suite software (version 5) yielded gene expression values as numeric values (signal intensities) and detection calls (“Present” or “Absent”) produced with different algorithms. A comparison between two arrays (tumor and control lung) resulted in the signal logarithm ratio (log 2 ratio) and a change call (“Increase” or “Decrease”) for expression level of each gene. Data from replicate samples were evaluated and compared using statistical analyses with the Affymetrix® Data Mining Tool 2 (DMT-2). To summarize the expression level (the signal values) for each transcript across the replicates were used the average and standard deviation statistics. Mean fold change values were calculated as the ratio of the average expression levels for each gene between two tissue sets. To determine significance of change in mean gene expression level between comparison groups the unpaired two-sided T-Test was used, with the p-value cutoff determined as 0.05. Additionally to parametric T-test, which uses for analysis numeric expression values (signal intensities), comparison ranking analysis was done to study the concordance between “Increase” or “Decrease” calls for tumors in one set. The results are shown as % of concordance between all comparisons, e.g. 16 analyses (4 tumors versus 4 controls) for tumors of median size and 12 (3 tumors versus 4 controls) for tumors of large size. Three pools of small tumors (1 mm) were compared with the 4 individual controls, resulting in 12 comparisons. The group of differently regulated genes in tumors was restricted to those genes, which were detected (“Present” call) in all samples of a tumor sets for the up regulated genes and in all control lungs for the down regulated genes. Further criteria were FC≧3, p-value in T-test ≦0,05 and 100% of “Increase” call in comparative ranking analysis for one or more sets of tumors versus control lungs for up regulated genes and FC ≦−3, p-value in T-test ≦0,05 and 100% of “Decrease” call accordingly for down regulated genes.

Gene Expression Studies by RT-PCR

The primer 3 software (Rozen S and Skaletsky H J, 2000) was used to design the primers. The amplification fragments spanning an intron were chosen to distinguish between amplification from contaminating DNA.

Total RNA was isolated with the Qiagen RNA purification kit according to the manufacturers instructions. Reverse transcription was carried out using Omniscript (Qiagen), Oligo-dT primers (InVitrogen) and RNasin (Promega) followed by PCR amplification (see above) with the following primer pairs: Ccnb1, fp: CAGTTGTGTGCCCAAGAAGA (SEQ ID NO 3); rp: TCCATTCACCGTTGTCAAGA (SEQ ID NO 4) (57°, 32 cycles). Cdc2a,fp: CTCGGCTCGTTACTCCACTCGAGCATCAAGAAAGAGGTCAAAGG (SEQ ID NO 5);rp: CCATTTTGCCAGAGATTCGT (SEQ ID NO 6) (56° C., 34 cycles). Stk6:fp:GCCCACTAGGAAAAGGGAAG (SEQ ID NO 7). rp: CGTTTGCCAACTCAGTGATG (SEQ ID NO 8) (56° C., 34 cycles). Cdk4, fp: AACTGATCGGGACATCAAGG (SEQ ID NO 9), rp:CACGGGTGTTGCGTATGTA (SEQ ID NO 10) (58° C., 30 cycles). Nek6, fp:TTGAGATGATGGATGCCAAA (SEQ ID NO 11), rp: AGCTGTGATGAACACGTTGG (SEQ ID NO 12) (56° C., 32 cycles). Prc1, fp: CATGATGCCGAGATTGTACG (SEQ ID NO 13), rp: CAGCCGATGTAATTCCCACT (SEQ ID NO 14) (57° C., 32 cycles). Birc5, fp: GAATCCTGCGTTTGAGTCGT (SEQ ID NO 15), rp:CAGGGGAGTGCTTTCTATGC (SEQ ID NO 16) (56° C., 38 cycles). Ddit3, fp:CTGCCTTTCACCTTGGAGAC (SEQ ID NO 17), rp: GGGCACTGACCACTCTGTTT (SEQ ID NO 18) (58° C., 34 cycles). Satb1 fp: GTGATGGCTCAGTTGCTGAA (SEQ ID NO 19), rp:CATAGCCCGAAGGTTTACCA (SEQ ID NO 20) (57° C., 32 cycles). Hey1 fp:GAGACCATCGAGGTGGAAAA (SEQ ID NO 21), rp: ACCCCAAACTCCGATAGTCC (SEQ ID NO 22) (58° C., 32 cycles); β-actin fp:GGCATTGTTACCAACTGGGACG (SEQ ID NO 23), rp: CTCTTTGATGTCACGCACGATTTC (SEQ ID NO 24) (65° C., 23 cycles). β-actin was used as a housekeeping gene because of the equal expression in all lung samples. PCR reactions were done using Taq Platinum PCR Super-Mix Kit (Invitrogen), and amplification products were separated on 1% agarose gels. A semiquantitative measurement of band intensities was done using Kodak 1D Image Analysis Software. The gene expression values in each samples were normalized to the beta-actin expression and then used to calculate mean expression values for the sets of tumors of various sizes and control lungs. The mean fold changes was computed as a ratio between mean gene expression values for tumor sets and control non transgenic lung as a baseline.

Western Blotting

Frozen lung tumors of about 0.5-1 cm from three different SPC/c-Myc transgenic mice and non transgenic lung from three control mice were thawed on ice. Protein extracts were prepared by sonications of the samples in 500 μl 2D-loading buffer with the following incubation with benzonase according to (Molloy et al., 1999) and stored at −80° C. 75 μg (c-Myc) or 100 μg (Hspa9a, Nek6) of total protein extracts were separated on 10% (Hspa9a), or 12.0% SDS-polyacrylamide gel (c-Myc, Nek6) and blotted onto PVDF membranes in 25 mM Tris and 190 mM Glycin at 4° C. for 2 h (10%, 12% gel) at 350 mA. Specific antibodies to detect the selected proteins were anti-c-Myc rabbit polyclonal (1:500), anti-GRP75 (Hspa9a) goat polyclonal (1:200) purchased from Santa Cruz and anti-NEK6 rabbit polyclonal (1:100) purchased from Abgent. Antigen-antibody complexes were visualized using the ECL detection system as recommended by the manufacturer NEN Life Science Products (PerkinElmer Life Science, Rodgau-Juegesheim, Germany) and recorded with Kodak IS 440 CF (Kodak, Biostep GmbH, Jahnsdorf, Germany).

Bioinformatic search for potential c-Myc-binding sites in 5′-UTR region of the differently expressed genes in tumors.

In order to identify c-Myc targets among genes differently regulated in lung adenocarcinoma were applied positional weight matrices (PWMs) that is the most widely used method for recognition of transcription factor binding sites (Quandt et al., 1995) in a bioinformatic analysis of putative promoter sequences of deregulated genes. For that purpose TRANSFAC® Professional rel. 8.3 database was used which contains the largest collection of weight matrices for eukaryotic transcription factors (Wingender et al., 2001). The UCSC Mouse Genome Browser was used to extract the putative promoter regions of the corresponding genes. The beginning of the first exon was considered to be a tentative TSS (transcription start site), and 2000 bp upstream and 100 bp downstream of TSS were extracted. For the analysis of the extracted sequences a search profile which included five PWMs for c-Myc transcription factor with accession numbers M01034, M00322, M00118, M00123, M00615 was applied. The MATCH™ algorithm calculating scores for the matches by using the so-called information vector (Kel et al., 2003) was employed. The matrix similarity cut-offs for the matrices were set to different values (1.0, 0.96, 0.93, 0.98, 0.995 respectively), so that the rate of false positive matches for each matrix was less than 1 per 10,000 bp.

Maintenance of the transgenic mouse line, sample collection and preparation, isolation of RNA, production of c-RNA, array hybridization and scanning, gene expression data analysis, bioinformatic search for c-Myc-binding sites in 5′-UTR region of differently expressed genes in tumors, gene expression studies by RT-PCR and Western blotting were performed as described in the first part of the study.

RT-PCRs were Performed with the Following Primer Pairs (fp;rp):

Shmt1, fp.GCAACTCTGAACCAGTGCAA (SEQ ID NO 25), rp:TGAGGATCCAGATGGTAGGC (SEQ ID NO 26) (56° C., 36 cycles). Satb1, fp:GTGATGGCTCAGTTGCTGAA (SEQ ID NO 27), rp:CATAGCCCGAAGGTTTACCA (SEQ ID NO 28) (57° C., 32 cycles); Arg1, fp: AAGCTGGTCTGCTGGAAAAA (SEQ ID NO 29), rp: CTGGTTGTCAGGGGAGTGTT (SEQ ID NO 30), (55° C., 36 cycles); Fasn, fp: TCTGCAGAGAAGCGAGCATA (SEQ ID NO 31), rp: GTCATTGGCCTCCTCAAAAA (SEQ ID NO 32) (55° C., 31 cycles); Srm, fp: GTCCAGTGCGAGATTGATGA (SEQ ID NO 33), rp: GCAGAAGTGCCTCATCTCCT (SEQ ID NO 34) (57° C., 32 cycles); Hk2, fp: GGTGGAGATGGAGAACCAGA (SEQ ID NO 35), rp: TCATTCACCACAGCCACAAT (SEQ ID NO 36) (55° C., 32 cycles); Tk1, fp: CCAGATCGCCCAGTACAAGT (SEQ ID NO 37), rp: GAAGCACTCCATGCACACAG (SEQ ID NO 38) (59° C., 34 cycles); Impdh2, fp: GGAAGTGGTTCCATCTGCAT (SEQ ID NO 39), rp: TGGCTGCTGAGATGTTTGTC (SEQ ID NO 40) (57° C., 32 cycles); Uck2, fp: CAGATCCCCGTGTACGACTT (SEQ ID NO 41), rp: GTCGGCACCTCTAGGAATGA (SEQ ID NO 42) (59° C., 32 cycles); Smarcc1, fp: TGATCCAAGTCGCTCAGTTG (SEQ ID NO 43), rp: TCACAGCACACACATCTCCA (SEQ ID NO 44) (57° C., 32 cycles); Npm3, fp: GGCTGCTTTAGCGTTCTTGA (SEQ ID NO 45), rp: AAGGTGACAGGTGGTTGGAG (SEQ ID NO 46) (57° C., 33 cycles); Slc19a1, fp: ATGTGCATGTCCTGTGGAGA (SEQ ID NO 47), rp: AGGGAAGACGCAATCTGAAA (SEQ ID NO 48) (55° C., 37 cycles); β-actin was used as a housekeeping gene, because its expression was found to be unchanged in lung tumors of SPC/c-Myc mice (microarray analyses) compared with non-transgenic lungs: fp:GGCATTGTTACCAACTGGGACG (SEQ ID NO 49), rp: CTCTTTGATGTCACGCACGATTTC (SEQ ID NO 50) (65° C., 23 cycles).

Western blotting was done using specific antibodies for selected proteins: anti-Arg1 (1:100), anti-cShtm (1:100), anti-HxkII goat polyclonal (1:100) and anti-Fasn rabbit polyclonal (1:100) antibodies purchased from Santa Cruz Biotechnology, Inc.

Maintenance of the transgenic mouse line, sample collection and preparation, isolation of RNA, production of c-RNA, array hybridization and scanning, gene expression data analysis, bioinformatics search for potential c-Myc-binding sites in 5′-UTR region of the differently expressed genes in tumors, gene expression studies by RT-PCR and Western blot were performed as described.

RT-PCRs were Performed with the Following Primer Pairs:

Ros1, fp: CAGTGGCACACGGTACAATC (SEQ ID NO 51); rp: CTCCGTGAAAGTCCAGCTTC (SEQ ID NO 52) (59°, 36 cycles). Ect2, fp: AGGACCTTCCATTCGAACCT (SEQ ID NO 53). rp: GACTCGGGTGTGTTGGAGAT (SEQ ID NO 54) (58° C., 34 cycles). Traf4: fp: CGGCTTCGACTACAAGTTCC (SEQ ID NO 55). rp: TAGGGCAGGGGACTACATTG (SEQ ID NO 56) (59° C., 34 cycles). Hgfac, fp: GCTTCCTGGGAAATGGTACA (SEQ ID NO 57), rp: CCTCTTGCCACAGGTAGGAC (SEQ ID NO 58) (58° C., 36 cycles). Adam19, fp: TTTACCGCTCCCTGAACATC (SEQ ID NO 59), rp: AGCAGTGTGCAGAATCATGG (SEQ ID NO 60) (57° C., 32 cycles). Bmp6, fp: TTCTTCAAGGTGAGCGAGGT (SEQ ID NO 61), rp: CTCGGGATTCATAAGGTGGA (SEQ ID NO 62) (57° C., 32 cycles). Acvrl1, fp: CCACAACGTGTCTCTGATGC (SEQ ID NO 63), rp: ACACTCTACCAGCGCAACCT (SEQ ID NO 64) (59° C., 32 cycles). Igfbp5, fp: CTACTCCCCCAAGGTCTTCC (SEQ ID NO 65), rp: TTGTCCACACACCAGCAGAT (SEQ ID NO 66) (57° C., 29 cycles). Sema3f, fp: GCTTCCAGCCACACCTAGAG (SEQ ID NO 67), rp: TACAGGTGTGTTCGGTTCCA (SEQ ID NO 68) (57° C., 34 cycles). Cav fp: GGGAACAGGGCAACATCTAC (SEQ ID NO 69), rp: GTGCAGGAAGGAGAGAATGG (SEQ ID NO 70) (59° C., 32 cycles). Cdh5 fp: CAATGACAACTTCCCCGTCT (SEQ ID NO 71), rp: TGTTTTTGCCTGAAGTGCTG (SEQ ID NO 72) (57° C., 29 cycles). β-actin was used as a housekeeping gene, because its expression was found to be unchanged in lung tumours of SPC/c-Myc mice (microarray analyses) compared with controls (non-transgenic lungs. The following primer pair was used: fp:GGCATTGTTACCAACTGGGACG (SEQ ID NO 23), rp: CTCTTTGATGTCACGCACGATTTC (SEQ ID NO 24) (65° C., 23 cycles).

Western blotting was done using specific antibodies for selected proteins: anti-Traf4 (1:100), anti-hepsin (1:100), anti-Alk1 (=Acvrl1) (1:100) goat polyclonal antibodies and anti-Igfbp6 (1:100) rabbit polyclonal antibody purchased from Santa Cruz Biotechnology, Inc., anti-claudin2 (1:500) rabbit polyclonal antibody purchased from Zymed Laboratories Inc., and Adam19 (1:1000) rabbit polyclonal antibody purchased from Cedarlane Laboratories.

The characteristics of the invention being disclosed in the preceding description, the subsequent drawings and claims can be of importance both singularly and in arbitrary combination for the implementation of the invention in its different embodiments.

The foregoing description of preferred embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form described, and many modifications and variations are possible in light of the teaching above. The embodiments were chosen and described in order to best explain the principles of the invention and its practical applications to thereby enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the claims.

Targeted overexpression of c-Myc to alveolar epithelium of mice induced multifocal non-invasive bronchioloalveolar carcinoma (BAC) and solitary invasive papillary lung adenocarcinoma (PLAC). Initially, we reported regulation of genes in papillary adenocarcinomas (Meier et al., part1-3). Here we report genes specifically regulated in BAC. Strikingly, only a few genes were commonly regulated in both subtypes of c-Myc-induced adenocarcinomas and included genes involved in cell cycle progression, e.g. Ccnb1, Cdc2a, Ect2, Shtm1 (induced), and Igf binding protein 2 with growth-modulating activity (repressed). The common regulation of these genes in BAC and PLACs is suggestive for their primary role in c-Myc-induced lung carcinogenesis. Notable, the majority of regulated genes differed for BAC and PLAC. Thus, activation of genes involved in angiogenesis like Angpt1, Calcrl, Edg3, and negative regulators of cell proliferation, e.g. Rasa1, Ptpns1, Mapk14, was found specifically in BAC. Repression of genes with growth-inhibitory functions like Tgfbr3, Lats2, Akap12 and induction of genes enhancing cell growth like Arg1, Hk2, Fasn, the proteases Adam19, Hgfac and hepsin were characteristic for PLAC. In conclusion, an identification of genes attributable to BAC and PLAC enables molecular finger printing of the two lung adenocarcinoma subtypes.

Lung cancer is a leading cause of death and a major public health problem throughout the world. Histologically adenocarcinomas differ and are subdivided into distinct subtypes on the basis of predominant cell morphology and growth pattern, i.e.—acinar, papillary, bronchioalveolar and mucinous adenocarcinomas (Notably, lung tumors induced by c-Myc were heterogenous and could be classified as either multicentric non-invasive bronchioloalveolar carcinoma (BAC) or solitary invasive papillary lung adenocarcinoma (PLAC) (Part1). Strikingly, regulation of genes differed mostly amongst BAC and PLAC when compared with control non-transgenic lung. Therefore, oncogenomics enabled an identification of genes specifically associated with either BAC or PLAC adenocarcinomas.

Results

Histology of Lung Tumors.

We specifically studied mice at the age of 8 months as they didn't contain macroscopically visible solid tumors (PLACs). This was confirmed by histopathology (FIG. 12). Note, the healthy lungs of non-transgenic animals served as a control. Furthermore, transgenic males and females were examined separately to explore sex differences in the development of lung tumors. Indeed, females developed tumors more quickly than males, because 75% of c-Myc transgenic lung was occupied by BAC in females and 60% in males at this age).

Oncogenomics of BAC

To study genetic events underlying the development of BAC we investigated genome wide transcript expression in the lungs of c-Myc-transgenic males and females at the age of 8 months. BAC growth was confirmed by histopathology. By use of the murine genome array 430 2.0 we identified 246 genes and ESTs differently expressed in transgenic lungs as compared with healthy non-transgenic lung (see Methods for the significant expression change criteria). Note 243 genes were up regulated but 3 genes were repressed. Gene expression changes were more prominent with females resulting in 241 significantly regulated genes. In males the same genes were regulated (FIG. 13) but only 5 genes reached the threshold for significance testing (Table 8). The lower gene expression values observed with males agreed well the lesser occupancy of BAC growth in the lungs of transgenic mice, e.g. 75% in females versus 60% in males which indicated a sex specific difference in tumor growth. We previously reported oncogenomic of PLACs using MGU74Av2 microarray (Affymetrix) and yielded 162 genes which were significantly activated and 301 genes which were repressed in these tumors (see Meier et al., 2006 Part1). To identify differences in the regulation of gene transcription in these two subtypes of lung adenocarcinomas we compared expression of significantly regulated genes identified in BAC with that in PLAC and vice versa. As we employed the latest MG430 2.0 microarray representing a greater number of mouse genes (˜14000) than MGU74Av2 (˜6000) used in our previous analysis, only ˜⅔ of regulated genes identified in BAC were represented on MGU74Av2 and could therefore be compared with PLAC. Though the thresholds for significantly regulated genes in PLAC were defined more stringent and less mouse genes are represented on the MGU74Av2 array, we identified much more significantly regulated genes in PLAC than in BAC. Altogether, 595 genes significantly regulated in BAC and/or PLAC were taken into comparison. To obtain a comprehensive comparison of gene expression in BAC and PLAC, a hierarchical clustering algorithm was applied to the sum of genes differently expressed in BAC and/or PLAC (FIG. 13). Fold changes of gene expression in BAC, in females and males, and PLAC, in small-, middle- and large-sized tumors were used for the comparison (FIG. 13). Strikingly, the gene expression profiles of BAC and PLAC were quite different, with only few genes being similarly regulated in these subtypes of lung adenocarcinoma when compared with control non-transgenic lung. Table 8 gives an account of genes differently expressed in BAC and/or in PLAC where they are divided into groups according to the specific regulation in BAC and PLAC. Only selected genes with inferred importance for tumor biology are given accompanied by fold changes of gene expression level and results of statistical analyses. Importantly, we identified only few genes which were similarly up regulated in BAC and PLAC. These genes included positive regulators of cell cycle like c-Myc itself, cyclin b1, Cdc2a and the proto-oncogene Ect2, the enzyme Shtm1 favouring DNA biosynthesis, the Hgf receptor c-Met, the adhesion molecule CD44, and the lipase related Pnliprp1 amongst others. Repression of Igfbp2 involved in modulation of survival signalling was also identified in both subtypes of tumors. In contrast, expression of most genes significantly induced in BAC was diminished in PLAC. Mostly, these genes were only slightly induced or not regulated or even repressed in PLAC when compared with non-transgenic control lung. These included, amongst others, genes coding for angiogenesis like Hif1, Angpt1, Calcrl, Edg3, as well as negative regulators of growth, e.g., Tgfbr3, Rasa1, Ptpns, Sgpl1 and the pro-apoptic Mapk14 (=p38MAPK). Among those genes with opposite regulation in their expression in BAC and PLAC we found the adhesion molecules like integrin alpha 8, Cd38 and cytoskeletal proteins utrofin and vinculin as notable examples. Numerous genes which we previously found to be regulated in PLAC (Part1-Part3) were not changed in BAC. For example, genes up regulated in PLACs which code for proteins in cell division and growth, e.g. Cks1, Cks2, Nek6, kinesins Kif11, Kif22 Kif4, arginase1, spermidine synthase, Impdh2, Uck2, hexokinase 2, lactate dehydrogenase, fatty acid synthase, were expressed in BAC similar to control lungs as were several translation initiation factors and cell and nuclear transporters. Besides, they included several proteases like Hgf activator, hepsin, Adam19, Thop1 and genes involved in pro-growth signaling like proto-oncogene Ros1, autocrine growth factor amphiregulin, Arhu, kininogen which therefore may contribute to the advanced tumor phenotype. Notably, among numerous genes repressed in PLAC and not changed in BAC we identified the growth inhibitory and tumor suppressors, e.g., Gas1, Septin4, Bmp6, Acvrl1, Akap12, Bnip2, Cav1, Capn2, Ddit3, Hey1, Igfbp6, Lats2, Lox, Ptprd, Sema 3f, several serpins and others (Part 1, 3). As was noted above, approximately one third of regulated genes identified in BAC have no reference to PLAC, because these genes were not represented on the MG U74Av2 as yet. These genes up regulated in BAC included, amongst others, mitotic kinase Pbk (PDZ binding kinase), Pdgfd, Tmem23, Eaf1, Amd1 and dCMP deaminase with important roles in cell proliferation and growth, Akt3 (v-akt murine thymoma viral oncogene homolog) involved in survival signaling, Sgk3 closely related to Akt and adducin3 mediating actin driven cell movement. Other genes like the mitotic spindle checkpoint Bub1b, the stress response protein Stch, Tlk1b involved in repair of genomic damage, the transcription factor Klf6 thought to be a tumor suppressor were induced in BAC likely as a part of a feed-back mechanism controlling cell growth and proliferation.

Discussion

1. Genes Activated in Either c-Myc-Induced Subtype of Lung Adenocarcinoma Code for Essential Regulators of Cell Proliferation

Comparison of gene expression profiles of c-Myc-induced BAC and PLAC revealed that only a limited number of genes were induced in both subtypes of lung adenocarcinoma when compared with healthy lung of non-transgenic mice (Table 8.1a, b). Notably, some of these genes were expressed at higher level in PLAC than in BAC (Table 8.1a) like the c-Myc transgene itself which was induced 3-5-fold in lungs with BAC and 23- to 58-fold in solid PLACLikely, expression of the oncogenic transgene may initially be kept under control, for instance, by recruitment of transcriptional repressors. In advanced stages of tumor growth the ability to suppress activation of the transgene may become less efficient because of impaired control mechanisms or/and autocrine stimulation by oncogene, e.g. c-Myc, of its own transcription through activation of appropriate signaling circuits giving rise to high level of transgene expression. Most genes whose expression was coherent with c-Myc expression coded for proteins essential for cell cycle progression. Thus, genes significantly induced in BAC and still stronger upregulated in PLAC were cyclin b1 (Ccnb1) and its kinase Cdc2a forming the complex which initiates the M phase of cell cycle. We additionally identified genes moderately increased in BAC and significantly induced in PLAC (Table 8, 1a) which are associated with cell cycle progression and proliferation. Identification of the above named genes point to molecular events induced by c-Myc to enhance cell proliferation in PLAC as compared with BAC. Besides, the PDZ-binding kinase (Pbk), which is a novel mitotic kinase up regulated in a variety of highly proliferative malignant cells, fetal tissues and hematologic malignancies (Simons-Evelyn et al., 2001), (Nandi et al., 2004), and dCMP deaminase (Dctd) involved in pyrimidine biosynthesis were significantly induced in BAC (Table 8, 1c) and may well represent new targets of c-Myc to stimulate cell division. Among those genes whose expression was significantly increased in BAC and similarly changed in PLAC (Table 8, 1b) we identified two receptors for growth factors, i.e. the c-Met proto-oncogene (Met) and the fibroblast growth factor receptor 2 (Fgfr2), the hyaluronic acid receptor Cd44 and syndecan1 (Sdc1) with roles in cell proliferation, cell migration and cell-matrix interactions in addition to the lipase-related Pnilprp1. This suggests that overexpression of members of a gene family of lipases may be of great importance for inappropriate cell proliferation through as yet unestablished mechanism. Increased expression of some genes with growth-, metastasis- and angiogenesis supressor functions like Ppp2r5c, Gja1, Rrm1 and Nedd 4 may be part of an anti-tumor response in lung cancer.

Thus, the comparison of genes differently expressed in BAC and PLAC proved to be instrumental in identifying particular genes with similar regulation in different subtypes of c-Myc-induced lung adenocarcinomas which suggest their primary role in c-Myc-trigged lung carcinogenesis. Tracing individual genes specific for tumor pathologies enables an identification of disease candidate genes.

Genes Up Regulated in BAC Contributing to Cell Growth

Several new genes which may prove to be essential in lung cancer development were identified in BAC by use of the MG 430 2.0 array (Table 8.1c). We identified regulation of the thymoma viral proto-oncogene 3 (Akt3), the platelet-derived growth factor, D (Pdgfd), the sphingomyelin synthase 1 (Tmem23=Sms1), the Adducin 3 (Add3), the S-adenosylmethionine decarboxylase 1 (Amd1), and the ELL associated factor 1 (Eaf1

2. Genes with Different Regulation in BAC and PLAC (See Also Table 8).

2.1 Genes Significantly Enhanced in BAC and Repressed in PLAC Included Those Coding for Angiogenesis, Growth Inhibitory Response and Adhesion

Among genes whose expression was strongly induced in BAC but repressed in PLAC (Table 8.2.1) we identified endothelial-specific growth factors angiopoietin 1 (Angpt1), (Zadeh et al., 2004), and the G-protein-coupled receptors, Edg3 (endothelial differentiation, sphingolipid G-protein-coupled receptor, 3) and Calcrl (calcitonin receptor-like receptor) which likely contribute to angiogenesis through increase of endothelial cell proliferation and survival (Fieber et al., 2006). Besides, genes whose expression was significantly increased in BAC and only slightly in PLAC (Table 8.1b) included hypoxia-inducible factor1 Hif1a. Hif1 plays an essential role in cellular and systemic homeostatic response to hypoxia, directly activating transcription of genes involved in energy metabolism, angiogenesis, vasomotor control, apoptosis, proliferation and matrix production, and can also be induced by hypoxia-independent factors like growth factor receptor (e.g. Egfr) signaling (Yudoh et al., 2005). Thus, Hif1a is a transcription factor that can directly transactivate genes important for the enhanced growth and metabolism. Activation of proangiogenic genes in BAC argues for the angiogenic capacity of c-Myc.

Furthermore, among genes activated in BAC but not in PLAC (Table 8.2.1) we observed regulation of transcripts coding for anti-growth capacities, e.g. the receptor for TGFβ family members Activation of the growth-inhibitory genes, may represent a part of a feed-back regulatory response to an inappropriate activation of proliferative signaling induced by constitutive c-Myc over expression. This feedback mechanism was absent in PLAC to foster aggressive tumor growth. Notably, genes oppositely regulated in BAC and PLAC, i.e., induced in BAC and repressed in PLAC, included cytoskeleton coding genes Utrn, Vcl and adhesion molecules Itga8, Cd38. Opposite regulation of these genes may be related to different growth pattern and invasive behaviour of BAC and PLAC.

Further, genes specifically regulated in BAC, but not in PLAC included the docking protein Gab1 utilized for Egfr- and c-Met signaling (Meng et al., 2005), the proto-oncogene Yes1 involved in integrin signaling (Klinghoffer et al., 1999), and the protease Adam 17 shown to mediate activation of Egfr in vivo in tumor development by nude mice (Borrell-Pages et al., 2003). Likewise, in BAC the apoptosis inhibitors Birc 4 (Tenev et al., 2005) and Api 5 (Kim et al., 2000), the proliferation-associated transferrin receptor 1 (Tfrc1) (Wang et al., 2005b) and the G-proteins Gna12 and Gna13 which interact with cadherins to release transcriptional activator β-catenin (Meigs et al., 2001) were significantly upregulated. Activation of the genes discussed herein represents unique features of BAC oncogenomics.

2.2 Genes Induced or Repressed in PLAC but not Regulated in BAC May Contribute to a More Aggressive and/or Invasive Phenotype of PLAC

We identified genes whose expression was significantly regulated in PLAC but unchanged in BAC (FIG. 13). A description of genes regulated in PLAC was reported in Meier et al. (Part 1-3). Here we focus on a selected group of genes to demonstrate their distinct and characteristic expression pattern in BAC or PLAC (Table 8, 2.2). Genes whose expression was significantly up regulated in PLAC included those involved in various aspects of cell growth like cell cycle progression, e.g. Cks1, Nek6, polyamine synthesis, e.g. Arg1 and Srm1, nucleotide synthesis, e.g. Impdh2, Uck2 and Rrm2, glycolysis, e.g. Hk2, Ldh, lipid metabolism, e.g. Fasn, transcription, e.g. Rpo1-3, protein synthesis e.g. Eif2b, nuclear transport e.g. Nol5a, and pro-growth signalling, e.g. Ros1 proto-oncogene, amphiregulin, Arhu and kininogen. Activation of such genes involved in cell proliferation and growth together with induction of several proteases, like Adam19, Hgf activator, hepsin, Thop1 and matrix metalloproteinase regulator lipocalin may well represent the molecular switches for more aggressive growth of PLAC. Another specific feature of PLACs was down regulation of numerous genes which, amongst others, included many growth-inhibitory and tumor suppressor genes (see Results and Table 8 and Meier et al. 2006 Part 1, 3). This suggests that suppression of these genes in PLAC was not the result of direct action of c-Myc, but rather represents secondary effect of genetic or epigenetic perturbations upon c-Myc over expression in advanced stages of tumor growth. Conclusion

In conclusion, we investigated the oncogenomics of distinct subtypes of lung adenocarcinoma in a c-Myc transgenic mouse model. The lung tumors arise from the same progenitor cells, e.g. alveolar epithelium. Few solitary PLACs were observed in mice at much later time points than multi-centric BAC which we found dispersed throughout the whole lung. Furthermore, genome wide expression profiling revealed more gene expression changes in PLAC than in BAC with a number of genes linked to cell cycle progression being stronger induced in PLAC than in BAC therefore indicating enhanced cell proliferation in PLAC. Besides, genes activated or repressed in PLAC could be linked to a more aggressive phenotype to enable invasive and metastatic growth. Collectively, these observations allowed us to hypothesize that BAC is an earlier stage and PLAC an advanced stage of the lung adenocarcinoma induced by c-Myc. Importantly, by genome wide expression profiling of different pathologies of c-Myc-induced lung tumors, e.g. non-invasive BAC and invasive PLAC, genes could be identified which represent, most likely, the molecular basis for malignant growth induced by c-Myc. Genes specifically regulated in BAC or PLAC can therefore be held responsible for the more aggressive phenotype observed with PLACs.

Methods

Maintenance of the Transgenic Mouse Line and Sample Collection

SPC/Myc transgenic mice (Ehrhardt et al., 2001) were maintained in the C57B1/6 background and identified as described previously (Meier et al., 2006, Part1). To study the transcriptome of BAC total RNA was isolated from the lungs of the transgenic mice at the age of 8 months. Normal lungs of non-transgenic animals served as a control. Mice were anesthetized by an overdose of CO₂ and the lung were isolated, one part of each lung being shock frozen in dry ice and stored at −80° for further gene expression studies and the other fixed in 4% formaldehyde and processed for histological investigation as described previously (Meier et al., 2006, Part1). 16 c-Myc transgenic males and 16 females were studied separately and compared with equivalent group of non-transgenic animals. For microarray analyses, equal amount of total RNA isolated from 4 individual mice were pooled, i.e. 4 pools of RNA, each pool combining RNAs from 4 mice, were analysed with microarrays per each animal group.

Isolation and microarray analysis of PLACs was described previously (Meier et al., 2006).

Transcriptome Analyses

Total RNA was isolated using the RNeasy total RNA isolation Mini Kit (Qiagen), pooled as described above, and a second clean-up of pooled RNA was done with the same RNeasy Kit. 8 mkg of total RNA were used to produce cRNA using One-Cycle Target Labeling and Control Reagents (Affymetrix) according to the Affymetrix Gene Chip® Expression Analysis Technical Manual (Santa Clara, Calif., 2005). Purified cRNA was quantified and checked for quality using the NanoDrop ND-1000 and the Agilent 2100 Bioanalyzer, then cleaved into fragments of 35-200 bases by metal-induced hydrolysis.

10 μg of biotinylated fragmented cRNA were hybridized onto the GeneChip® Mouse Genome 430 2.0 array from Affymetrix, washed and stained according to the manufacturer's recommendation. The arrays were scanned using the GeneChip® Scanner 3000. Scanned images were analyzed with the GeneChip® Operating Software (GCOS), each image being scaled to the same all probe set intensity for proper comparison between chips. Default parameters provided in the Affymetrix data analysis software package were applied for analysis.

Data Analysis

Identification of Significantly Regulated Genes

Gene expression data analysis for a single microarrays and a comparison between two arrays were performed using GCOS software. It applies two different algorithms to produce for each gene on the array numeric expression value and detection call (Present, Absent) and, by comparison of two samples (=arrays), additionally a signal logarithm ratio and a change call (“Increase”, “Decrease” or “No Change”).Statistical evaluation of replicate data was done with the Affymetrix® Data Mining Tool 3.1 (DMT). Fold change values were calculated as the ratio of the average expression levels for each gene between two tissue sets, i.e. BAC and normal lung, for males and females separately. To determine significance of gene expression changes in BAC compared with normal lung the unpaired two-sided T-Test with the p-value=0.05 as a threshold of significance was done using numeric gene expression values. Additionally, comparison ranking analysis was done to determine the concordance between change calls (“Increase” or “Decrease”) for regulated genes in BAC derived from different mice. The results are shown in Table 8 as % of change calls in 16 analyses (each of 4 tumor pools versus each of 4 normal lung pools). The thresholds for significantly up/down regulated genes in BAC were defined as a mean FC ≧=2,5/<=−2,5, a p-value in T-test <=0,05 and number of change calls >=87,5% (i.e. more than 14 out of 16) in comparative ranking in the group of transgenic females or males. The criteria for genes significantly up/down regulated in PLAC were defined more stringent and represented FC≧3 or <−3, p-value in T-test ≦0,05 and 100% of “Increase”/“Decrease” calls in comparative ranking in at least one group of tumors of different size, i.e. small- (n=3), middle- (n=4) and large-sized tumors (n=3), in comparison to normal lung (n=4) (Meier et al., 2006. Part1). Besides; both in BAC and PLAC analyses, we restricted our consideration for those up or down regulated genes, which were detected (“Present” call) correspondingly in all tumor or in all control lung replicates. In Table 8 the statistical values for all 10 PLACs are reported, whereas by hierarchical clustering the data for PLACs of different sizes were analysed separately (see below).

Hierarchical Gene Cluster Analysis,

To get an overall view of similarity and differences in regulation of gene expression in c-Myc-induced BAC and PLAC hierarchical gene cluster analysis implemented in ArrayTrack software (Tong et al., 2004) was done. Ward fusion algorithm with Euclidean distance was applied to the log transformed (with the base 2) ratios of gene expression values. The list of analysed genes combined all 597 genes significantly deregulated in BAC, males or females, and in PLAC, in small-middle- and large-sized tumors.

TABLE 1 Statistic of genes differently regulated in SPC/c-Myc- induced lung tumors Number of genes expressed in 4 control lung samples: 5269 Number of genes expressed in 10 lung tumor samples: 4706 Up regulated genes UP-regulated genes in tumors which promoters S > 70 & FC > 3 & 100% P, I & p < 0.05 in contain c-Myc one or more sets of tumors binding sites all up regulated genes in tumors 162 67 41.4% known direct c-Myc-targets (T) 32 20 62.5% relatives of known direct 15 5 33.3% c-Myc-targets (rT) known c- Myc- responsive genes (R) 16 8   50% relatives of known c-Myc-responsive 9 2 22.2% genes (rR) new c-Myc -responsive genes 90 32 35.6% DOWN-regulated genes in tumors Down regulated 100% P & S > 70 in control lungs & genes which FC < −3 & p < 0.05 & 100% D in promoters contain one or more sets of tumors c-Myc binding sites all down regulated genes in tumors 301 52 17.2% known direct c-Myc-targets (T) 5 1   20% relatives of known direct 16 2 12.5% c-Myc-targets (rT) known c- Myc- responsive genes (R) 24 5 20.8% relatives of known c-Myc-responsive 17 4 23.5% genes (rR) new c-Myc responsive genes 239 40 16.7% S—expression signal intensity FC—mean fold change of expression values in tumors compared with the control lungs P—“Present” detection call in Affymetrix microarray analysis I—“Increase” change call in Affymetrix microarray analysis D—“Decrease” change call in Affymetrix microarray analysis p—p-value in T-test between sets of tumors and control lungs sets of tumors: tumors of small, middle and large size

TABLE 2 Gene expression signatures in c-Myc-induced lung papillary adenocarcinoma: genes involved in cell proliferation, growth and apoptosis: (a)- up regulated genes; (b) down regulated genes p-value in T-test % compared to FC Increase/Decrease¹ control lung Gene size of tumors size of tumors size of tumors ACC Symbol Gene Title N small middle large small middle large small middle large (a) Up regulated genes in C-Myc induced lung tumors Cell proliferation: Cell cycle promoting genes G1/S AA791962 Cdk4 cyclin-dependent kinase 4 T, R 2 2.7 2.1 2.9 100 100 100 0.00 0.10 0.02 AI849928 Ccnd1 cyclinD1 R 2 1.6 1.5 1.4 100 56 58 0.00 0.05 0.04 X72310 Tfdp1 transcription factor Dp 1 R 3.3 2.5 3.1 100 88 100 0.01 0.06 0.02 G2/M X64713 Ccnb1 cyclin B1 T, R 22.2  9.4 12.8  100 88 66 0.03 0.16 0.33 X66032 Ccnb2 cyclin B2 rT 7.6 5.1 4.2 100 69 58 0.03 0.11 0.24 M38724 Cdc2a cell division cycle 2 homolog A R 18.0  10.6 10.5  100 50 33 0.01 0.06 0.19 (S. pombe) AB025409 Cks1 CDC28 protein kinase 1 rT, 9.1 6.6 7.0 100 100 100 0.01 0.03 0.02 rR AA681998 Cks2 CDC28 protein kinase regulatory T, R 10.1  6.0 5.8 100 100 100 0.04 0.08 0.24 subunit 2 AW061324 Cdc20 cell division cycle 20 homolog 8.3 5.1 4.5 100 94 91 0.05 0.14 0.19 (S. cerevisiae) U80932 Stk6 serine/threonine kinase 6 6.4 3.4 4.2 100 75 66 0.01 0.08 0.30 AI846534 Nek6 NIMA (never in mitosis gene a)- 3.1 3.2 3.5 100 100 100 0.00 0.01 0.01 related expressed kinase 6 AA856349 Prc1 protein regulator of cytokinesis 1 1 6.0 3.0 4.2 100 44 66 0.00 0.04 0.23 L11316 Ect2 ect2 oncogene 10.3  4.6 4.4 100 94 100 0.02 0.12 0.20 X82786 Mki67 antigen identified by monoclonal 21.6  8.0 11.6  100 75 66 0.05 0.12 0.26 antibody Ki 67 AJ223293 Kif11 kinesin family member 11 T 1 4.3 2.9 3.3 100 50 33 0.02 0.14 0.30 D12646 Kif4 kinesin family member 4 rT 3.2 2.3 2.3 100 69 58 0.05 0.05 0.22 AI131895 Kif22 kinesin family member 22 rT 8.1 5.0 5.2 100 75 91 0.01 0.05 0.18 Inhibitors of antiproliferative signaling AB013819 Birc5 baculoviral IAP repeat-containing 5 8.6 4.5 6.0 100 50 66 0.01 0.15 0.32 D17666 Hspa9a heat shock protein, A T 2 2.5 2.5 3.3 100 88 100 0.01 0.05 0.02 Apoptosis AF041377 Cideb cell death-inducing DNA 11.2  14.9 11.6  100 100 100 0.01 0.06 0.10 fragmentation factor, alpha subunit- like effector B AB021961 Trp53 transformation related protein 53 T, R 1 2.9 1.9 3.1 100 44 100 0.01 0.13 0.03 AF076482 Pglyrp peptidoglycan recognition protein 1.9 3.3 3.1 83 100 100 0.03 0.04 0.04 Transcriptions factors M32057 Zfp239 zinc finger protein 239 4.6 3.7 6.4 91 44 100 0.00 0.21 0.03 AF049702 Elf5 E74-like factor 5 1 5.6 6.1 6.1 100 100 100 0.00 0.02 0.03 AB008174 Tcf2 transcription factor 2 rR 3.0 2.8 3.2 100 100 100 0.00 0.00 0.00 M62362 Cebpa CCAAT/enhancer binding protein T, R 2 3.0 2.6 3.2 100 100 100 0.00 0.00 0.01 (C/EBP), alpha (b) Down regulated genes in c-Myc induced lung tumors Inhibitors of growth: Cell cycle control U19597 Cdkn2d cyclin-dependent kinase inhibitor −1.9  −4.1  75 83 0.08 0.03 2D (p19,inhibits CDK4) AI849416 Lats2 large tumor suppressor 2 −3.3  −3.3 −5.8  100 100 100 0.00 0.00 0.00 Apoptosis: p53-mediated pathway U20344 Klf4 Kruppel-like factor 4 (gut) R −8.8  −8.7 −13.2  100 100 100 0.01 0.01 0.01 AJ243895 Hey1 hairy/enhancer-of-split related −4.9  −3.7 −5.1  100 93 100 0.00 0.00 0.00 with YRPW motif 1 U06924 Stat1 signal transducer and activator of −2.6  −3.2 −4.8  100 100 100 0.01 0.01 0.01 transcription 1 X65128 Gas1 growth arrest specific 1 R −7.3  −5.8 −5.7  100 100 100 0.00 0.00 0.00 negative regulators of Bcl-2-mediated anti-apoptic pathway AF035207 Bnip2 BCL2/adenovirus E1B 19 kDa- −2.4  −3.0 −3.4  100 100 100 0.00 0.00 0.00 interacting protein 1, NIP2 D38117 Capn2 calpain 2 −3.0  −3.0 −3.8  100 100 100 0.02 0.03 0.02 U73478 Anp32a acidic (leucine-rich) nuclear −1.7  −3.6 −3.8  66 100 100 0.29 0.00 0.01 phosphoprotein 32 family, member A Lats2 (s. cell cycle) X67083 Ddit3 DNA-damage inducible transcript 3 R −2.3  −3.5 −6.2  100 100 100 0.03 0.01 0.01 Transcription factors X74134 Nr2f1 nuclear receptor subfamily 2, R −3.1  −3.1 −3.1  100 87 100 0.00 0.00 0.00 group F, member 1 AF010133 Madh6 MAD homolog 6 (Drosophila) −4.8  −5.8 −6.9  100 100 100 0.01 0.01 0.01 L35949 Foxf1a forkhead box F1a 1 −9.3  −12.6 −19.4  100 100 100 0.00 0.00 0.00 AW121328 Tbx3 T-box 3 2 −10.6  −10.6 −10.6  100 100 100 0.00 0.00 0.00 AF035717 Tcf21 transcription factor 21 −9.4  −12.7 −31.6  100 100 100 0.01 0.01 0.01 X55123 Gata3 GATA binding protein 3 −5.7  −5.7 −5.7  100 93 100 0.01 0.01 0.01 X94127 Sox2 SRY-box containing gene 2 −16.4  1.0 −6.1  100 62 100 0.00 0.99 0.01 AF041847 Crap cardiac responsive adriamycin protein −19.6  −27.6 −27.6  100 100 100 0.03 0.03 0.03 J03776 Trim30 tripartite motif protein 30 −3.4  −3.4 −3.4  100 93 100 0.02 0.01 0.01 AI853712 Klf7 Kruppel-like factor 7 (ubiquitous) 2 −3.1  −2.6 −3.4  100 93 100 0.00 0.01 0.01 D49473 Sox17 SRY-box containing gene 17 −5.8  −6.9 −8.6  100 100 100 0.01 0.01 0.00 L35032 Sox18 SRY-box containing gene 18 −5.5  −6.6 −6.3  100 100 100 0.00 0.00 0.00 U33629 Meis1 myeloid ecotropic viral integration −4.6  −4.6 −4.6  100 100 100 0.00 0.00 0.00 site 1 Y12293 Foxf2 forkhead box F2 −5.7  −5.7 −5.7  100 100 100 0.00 0.00 0.00 T - known c-Myc-target R - known c-Myc responsive gene rT - relative of known c-Myc-target rR - relative of known c-Myc-responsive gene N - number of potential c-Myc binding sites in promotor sequence ¹% Increase/Decrease - concordance of change calls in the pairwise comparisons (each tumor to each normal lung sample), by which genes are up or down regulated respectively

TABLE 3 Fold changes of gene expression in lung tumors determined in RT-PCR and microarray analysis Mean FC size of tumors Gene Method small middle large Cdk4 RT-PCR 3.0 2.0 1.4 Affymetrix 2.7 2.1 2.9 Stk6 RT-PCR 6.6 3.1 4.1 Affymetrix 6.4 3.4 4.2 Nek6 RT-PCR 3.0 2.6 2.9 Affymetrix 3.1 3.2 3.5 Ccnb1 RT-PCR 9.5 4.0 5.4 Affymetrix 22.2 9.4 12.8 Cdc2a RT-PCR 13.3 6.5 8.3 Affymetrix 18.0 10.6 10.5 Prc1 RT-PCR 8.0 5.1 5.5 Affymetrix 6.0 3.0 4.2 Birc5 RT-PCR 10.1 4.8 4.9 Affymetrix 8.6 4.5 6.0 Hey1 RT-PCR A −1.9 A Affymetrix A −3.7 A Ddit3 RT-PCR −4.0 −3.5 −5.9 Affymetrix A A A A—“Absent” - no expression was detected

TABLE 4 Gene expression signature in c-Myc-induced lung papillary adenocarcinoma: differently expressed genes* involved in stimulation of cell proliferation and growth p-value in T-test % compared to FC Increase/Decrease** control lung Gene size of tumors size of tumors size of tumors ACC Symbol Gene Title N small middle large small middle large small middle large Cell growth: metabolism, glycolysis U51805 Arg1 arginase 1, liver 6.1 10.8 14.8   94 100 100 0.01 0.09 0.27 X13135 Fasn fatty acid synthase T, R 2 3.1 2.9 3.1 100 100 100 0.00 0.03 0.09 AB033887 Facl4 fatty acid-Coenzyme A ligase, rR 2.7 3.2 3.2 91 88 100 0.09 0.02 0.00 long chain 4 Z67748 Srm spermidine synthase T, R 5 4.6 3.4 5.3 100 88 100 0.00 0.15 0.05 AA913994 Shmt1 serine hydroxymethyl transferase 1 T, R 4 7.5 7.3 9.4 100 100 100 0.01 0.04 0.04 (soluble) AI841389 Eno1 enolase 1, alpha non-neuron T, R 2.3 2.3 3.5 100 100 100 0.00 0.01 0.05 M17516 Ldh1 lactate dehydrogenase 1, A chain T, R 3.4 3.3 3.7 100 100 100 0.01 0.01 0.03 Y11666 Hk2 hexokinase 2 R 2.1 3.6 4.8 100 100 100 0.01 0.03 0.02 J05277 Hk1 hexokinase 1 2 2.8 2.0 3.2 100 75 100 0.01 0.13 0.00 M32599 Gapd glyceraldehyde-3-phosphate 1 3.0 2.9 3.5 100 100 100 0.01 0.00 0.04 dehydrogenase AF058956 Suclg2 succinate-Coenzyme A ligase, GDP- 2.2 2.2 3.0 100 94 100 0.00 0.01 0.02 forming, beta subunit M14220 Gpi1 glucose phosphate isomerase 1 R 4.2 2.0 2.7 100 50 33 0.05 0.29 0.36 L31777 Tpi triosephosphate isomerase 1 2.2 2.8 3.4 100 100 100 0.01 0.00 0.01 AW123026 Gnpnat1 glucosamine-phosphate 1 2.8 2.7 3.5 100 94 100 0.04 0.04 0.02 N-acetyltransferase 1 AB030316 Pign phosphatidylinositol glycan, class N 2.8 1.8 3.2 50 50 100 0.03 0.29 0.00 nucleotide biosynthesis, DNA metabolism M13352 Tyms thymidylate synthase T, R 3.7 2.2 2.3 100 63 75 0.00 0.12 0.20 X60980 Tk1 thymidine kinase 1 T, R 4.2 2.5 2.9 100 19 33 0.00 0.05 0.22 U20892 Gart phosphoribosylglycinamide R 1 3.0 2.6 3.4 100 94 100 0.01 0.06 0.04 formyltransferase M33934 Impdh2 inosine 5′-phosphate dehydrogenase 2 R 2 4.5 3.5 4.5 100 100 100 0.01 0.03 0.02 AI850362 Uck2- uridine-cytidine kinase 2 1 4.4 2.8 4.5 100 100 100 0.00 0.06 0.02 pending U60318 Mre11a meiotic recombination 11 homolog A 3.6 2.5 3.2 100 50 100 0.02 0.02 0.01

 

U01915 Top2a topoisomerase (DNA) II alpha 10.6 4.8 7.2 100 50 33 0.05 0.07 0.27 L32836 Ahcy S-adenosylhomocysteine hydrolase R 3.1 2.3 3.0 100 88 100 0.00 0.03 0.07 AI841645 Ard1 N-acetyltransferase ARD1 homolog 3.3 2.7 2.9 100 100 100 0.00 0.03 0.02 (S. cerevisiae) X67668 Hmgb2 high mobility group box 2 4.3 2.4 2.7 100 75 75 0.01 0.14 0.24 D90374 Apex1 apurinic/apyrimidinic endonuclease 1 T, R 2 6.6 4.0 5.9 100 100 100 0.00 0.06 0.00 X66323 Xrcc5 X-ray repair complementing defective 3.5 3.0 3.3 100 100 100 0.00 0.00 0.02 repair in Chinese hamster cells 5 K02927 Rrm1 ribonucleotide reductase M1 3.8 2.2 3.2 100 25 33 0.00 0.12 0.31 M14223 Rrm2 ribonucleotide reductase M2 5.8 2.7 3.8 100 6 66 0.00 0.06 0.24 AW122092 Rfc4 replication factor C (activator 1) 4 R 4.7 3.1 3.6 100 75 100 0.01 0.05 0.03 M38700 G22p1 thyroid autoantigen T 4.1 3.2 4.0 100 94 100 0.00 0.09 0.00 U25691 Hells helicase, lymphoid specific R 1 3.3 1.6 3.1 100 75 100 0.00 0.01 0.10 Chromatin structure U85614 Smarcc1 SWI/SNF related, matrix associated, 1 3.0 2.2 2.6 100 81 100 0.02 0.02 0.00 actin dependent regulator of chromatin, subfamily c, member 1 AI851599 H1fx H1 histone family, member X 4.8 5.8 6.4 100 8 24 0.03 0.10 0.14 M35153 Lmnb1 lamin B1 3.8 2.1 2.7 100 56 91 0.01 0.07 0.07 RNA processing, ribosomal biogenesis AI853173 Rpo1-3 RNA polymerase 1-3 rR 3.2 2.7 3.0 100 81 100 0.00 0.04 0.00 AI838709 Spnr spermatid perinuclear RNA binding 6.1 7.1 5.3 100 100 100 0.01 0.02 0.02 protein AW121447 No15a nucleolar protein 5A R 2 5.4 3.7 5.2 100 81 100 0.01 0.11 0.02 AA656775 Rrs1 RRS1 ribosome biogenesis regulator R 3 4.2 2.9 4.6 100 75 100 0.00 0.08 0.03 homolog (S. cerevisiae) X07699 Ncl nucleolin T, R 4 3.5 2.7 3.5 100 81 100 0.01 0.03 0.02 M33212 Npm1 nucleophosmin 1 T, R 3 3.7 3.1 4.0 100 100 100 0.00 0.02 0.01 U64450 Npm3 nucleoplasmin 3 2 6.0 5.5 7.4 100 94 100 0.01 0.05 0.04 AI183202 Hnrpa1 heterogeneous nuclear T, R 2 2.7 2.1 3.0 100 75 100 0.00 0.04 0.02 ribonucleoprotein A1 Z22593 Fbl fibrillarin R 1 3.2 2.8 3.6 100 94 100 0.00 0.02 0.01 AF053232 Nol5 nucleolar protein 5 1 7.7 4.8 6.6 100 100 100 0.00 0.04 0.02 AW060597 Snrpg small nuclear ribonucleoprotein rT 2.7 2.3 3.2 100 81 100 0.02 0.10 0.04 polypeptide G AA684508 Rnu22 RNA, U22 small nucleolar rT 4.8 3.1 3.8 100 69 83 0.00 0.03 0.02 AI853113 Cpsf5 cleavage and polyadenylation specific rT 2.9 2.6 3.1 100 88 100 0.01 0.02 0.03 factor 5* AA656757 Pabpc4 poly A binding protein, cytoplasmic 4 T 2 5.2 4.0 5.8 100 94 100 0.00 0.04 0.03 AI851198 Nola1 nucleolar protein family A, member 1 1 4.6 3.6 4.9 100 88 100 0.01 0.07 0.02 (H/ACA small nucleolar RNPs) AI852665 Mki67ip Mki67 (FHA domain) interacting 5.2 3.3 3.6 100 88 100 0.01 0.11 0.01 nucleolar phosphoprotein AA674812 Ppan peter pan homolog (Drosophila) 1 4.0 2.6 3.7 100 75 100 0.01 0.06 0.00 AI852608 Rnac- RNA cyclase homolog 2 3.6 2.7 4.0 100 88 100 0.02 0.04 0.00 pending AI845664 Grwd1 glutamate-rich WD repeat containing 1 3.8 2.1 3.2 91 44 100 0.00 0.16 0.02 protein synthesis and metabolism AW122030 Psat1 phosphoserine aminotransferase 1 T, R 7.2 5.3 5.5 100 100 100 0.02 0.03 0.03 U12403 Rpl10a ribosomal protein L10A T, R 1 2.6 2.2 3.2 100 81 100 0.00 0.05 0.03 X51528 Rpl13a ribosomal protein L13a T, R 6.3 4.3 5.0 100 100 100 0.00 0.02 0.04 X05021 Rpl27a ribosomal protein L27a T, R 1 2.9 2.4 3.3 100 75 100 0.00 0.05 0.02 AW060951 Bzw2 basic leucine zipper and W2 domains 2 rT 2 5.4 4.1 6.2 100 81 100 0.00 0.10 0.00 AI839363 Eif3s6 eukaryotic translation initiation factor 3, T, R 1 4.1 3.2 4.4 100 75 100 0.00 0.09 0.01 subunit 6 AV170770 Cct5 chaperonin subunit 5 (epsilon) T, R 1 4.1 3.4 3.6 100 75 100 0.00 0.05 0.00 AW122851 Fkbp11 FK506 binding protein 11 rT 1 7.9 7.5 9.1 100 75 100 0.00 0.12 0.05 AI840579 Srr serine racemase 4.5 3.6 5.0 100 100 100 0.00 0.04 0.02 AW124889 Pycs pyrroline-5-carboxylate synthetase 1 3.6 2.0 4.4 100 50 100 0.00 0.04 0.01 (glutamate gamma-semialdehyde synthetase) AW046590 Pcbd 6-pyruvoyl-tetrahydropterin 5.0 4.3 5.0 100 100 100 0.01 0.01 0.03 synthase/dimerization cofactor of hepatocyte nuclear factor 1 alpha (TCF1) AI840436 Mrps5 mitochondrial ribosomal protein S5 2.4 2.2 3.3 100 50 100 0.09 0.01 0.00 X06406 Lamr1 laminin receptor 1 (ribosomal 2 2.7 2.2 3.1 83 75 100 0.01 0.04 0.01 protein SA) AW124432 Mrpl12 mitochondrial ribosomal protein L12 4.0 3.5 3.2 100 88 100 0.00 0.07 0.01 AW045418 Rpl44 ribosomal protein L44 1 2.6 2.1 3.1 100 75 100 0.00 0.05 0.01 AW120719 Eif2b1 eukaryotic translation initiation 4.3 3.2 3.3 100 94 100 0.00 0.03 0.00 factor 2B, subunit 1 (alpha) transport AW125446 Golph2 golgi phosphoprotein 2 rT 4.1 5.1 4.6 100 100 100 0.01 0.00 0.04 AW122428 Timm10 translocase of inner mitochondrial rR 1 5.7 3.3 4.2 100 50 100 0.01 0.14 0.03 membrane 10 homolog (yeast) AA655369 Timm8a translocase of inner mitochondrial rR 2 5.8 4.5 5.7 100 100 100 0.00 0.07 0.01 membrane 8 homolog a (yeast) AF043249 Tomm40 translocase of outer mitochondrial 2.7 2.3 3.2 100 38 100 0.00 0.00 0.00 membrane 40 homolog (yeast) AI846308 Sfxn1 sideroflexin 1 R 1 2.9 2.4 3.1 100 88 100 0.00 0.05 0.00 L23755 Slc19a1 solute carrier family 19 (sodium/ T 1 10.4 6.6 11.1  100 75 100 0.02 0.04 0.03 hydrogen exchanger), member 1 AI846682 Slc15a2 solute carrier family 15 (H+/peptide 2.1 2.7 3.6 66 88 100 0.21 0.02 0.03 transporter), member 2 AI594427 Slc4a7 solute carrier family 4, sodium 2.5 2.3 3.1 100 88 100 0.00 0.04 0.00 bicarbonate cotransporter, member 7 AF020195 Slc4a4 solute carrier family 4 (anion 2.1 4.2 4.3 91 100 100 0.01 0.09 0.05 exchanger), member 4 U88623 Aqp4 aquaporin 4 rR 7.1 5.0 4.9 100 75 66 0.02 0.08 0.25 AI846319 Rangnrf- RAN guanine nucleotide release factor 1 4.2 3.6 5.7 100 50 91 0.00 0.05 0.00 pending D55720 Kpna2 karyopherin (importin) alpha 2 rT 3.2 1.8 1.7 100 56 41 0.03 0.17 0.36 AI847564 Kpnb3 karyopherin (importin) beta 3 1 3.9 2.7 3.8 100 94 100 0.02 0.03 0.01 AW212243 Ipo4 importin 4 3.6 2.8 3.7 100 81 100 0.00 0.01 0.00 X61399 Mlp MARCKS-like protein 1 3.3 2.5 4.6 100 94 100 0.01 0.02 0.03 M60348 Abcb1b ATP-binding cassette, sub-family B rT 1 2.0 1.6 3.2 83 50 100 0.02 0.28 0.00 (MDR/TAP), member 1B AF099988 Stk39 serine/threonine kinase 39, STE20/SPS1 1 3.3 3.3 4.3 100 100 100 0.01 0.01 0.02 homolog (yeast) Chromatin remodelling U05252 Satb1 special AT-rich sequence binding −5.4 −5.6 −7.0  100 100 100 0.01 0.01 0.00 protein 1 U73478 Anp32a acidic (leucine-rich) nuclear −1.7 −3.6 −3.8  66 100 100 0.29 0.00 0.01 phosphoprotein 32 family, member A X05862 Hist1h2bc histone 1, H2bc rT −3.0 −3.0 −4.9  100 100 100 0.00 0.000 0.00 *Criteria for significant expression changes were as follows: a gene should be expressed (“Present” call) in all tumor replicates of one set or in all control non-transgenic lungs for up- and down-regulated genes respectively and have a mean fold change (FC) ≧ 3, p-value in T-test ≦0.05 and 100% “Increase” call in comparative ranking analysis for up regulated genes and FC ≦ −3, p-value in T-test ≦0.05 and 100% “Decrease” for down regulated genes T - known c-Myc-target R - known c-Myc responsive gene rT - relative of known c-Myc-target rR - relative of known c-Myc-responsive gene N - number of potential c-Myc binding sites in promotor sequence **% Increase/Decrease - concordance of change calls in the pairwise comparisons (each tumor to each normal lung sample), by which genes are up or down regulated respectively Bold gene cell indicates that gene was not detected in all control lungs (for up regulated genes). Bold row indicates that gene was not detected in all control lungs and was expressed in all tumor samples (for up regulated genes) and inversely for down regulated genes

TABLE 5 Fold changes of gene expression in lung tumors determined by RT-PCR and microarray analysis Mean FC size of tumors Gene Method small middle large Shtm1 RT-PCR 3.1 2.4 2.7 Affymetrix 7.5 7.3 9.4 Npm3 RT-PCR 3.8 2.5 2.8 Affymetrix 6.0 5.5 7.4 Tk1 RT-PCR 8.7 3.9 4.4 Affymetrix 4.2 2.5 2.9 Uck2 RT-PCR 3.1 1.9 2.4 Affymetrix 4.4 2.8 4.5 Impdh2 RT-PCR 2.8 2.1 2.4 Affymetrix 4.5 3.5 4.5 Srm1 RT-PCR 5.5 3.5 4.1 Affymetrix 4.6 3.4 5.3 Arg1 RT-PCR 4.3 4.0 4.6 Affymetrix 6.1 10.8 14.8 Fasn RT-PCR 1.9 1.5 1.7 Affymetrix 3.1 2.9 3.1 Hk2 RT-PCR 1.5 2.1 2.4 Affymetrix 2.1 3.6 4.8 Slc19a1 RT-PCR 4.3 3.8 4.7 Affymetrix 10.4 6.6 11.1 Smarcc1 RT-PCR 1.8 1.4 1.7 Affymetrix 3.0 2.2 2.6 Satb1 RT-PCR A A A Affymetrix −5.4 −5.6 −7.0 A—“Absent” - no expression was detected

TABLE 6 Gene expression profiles in c-Myc-induced lung papillary adenocarcinoma: differently expressed genes involved in extracellular control of cell proliferation, adhesion, invasion and angiogenesis: (a) - up regulated genes; (b) down regulated genes p-value in T-test FC % Increase*/ compared to size of Decrease control lung Gene tumors size of tumors size of tumors ACC Symbol Gene Title N small middle large small middle large small middle large (a) Up regulated genes in C-Myc induced lung tumors Activation of growth factors signaling through tyrosine knase receptors L41352 Areg amphiregulin  5.1 7.7  8.3 100 100 66 0.01 0.07 0.18 AI467390 Map2k1 mitogen activated protein kinase rT 1  2.7 2.7  3.6 100 100 100 0.00 0.01 0.01 kinase 1* L00039 Myc myelocytomatosis oncogene 51.0 23.4 58.6 100 100 100 0.01 0.05 0.04 U15443 Ros1 Ros1 proto-oncogene  9.3 13.3 21.9 100 75 100 0.02 0.24 0.19 Activation of Wnt/beta-catenin signaling AW121294 Arhu ras homolog gene family, 1  4.0 4.2  3.8 100 44 66 0.00 0.21 0.18 member U AI646638 Frat2 frequently rearranged in  5.0 4.6  5.9 100 75 100 0.02 0.04 0.00 advanced T-cell lymphomas 2 Decrease of death receptor signaling AV109962 Traf4 Tnf receptor associated factor 4 1  4.3 5.0  4.5 100 100 100 0.01 0.00 0.03 Cell junctions AF072128 Cldn2 claudin 2 20.7 24.0 45.3 33 50 100 0.12 0.06 0.03 X63099 Gjb3 gap junction membrane channel 1  8.7 7.5  8.8 100 100 100 0.00 0.01 0.02 protein beta 3 M63801 Gja1 gap junction membrane channel 2  3.2 1.8  2.9 100 75 100 0.00 0.03 0.05 protein alpha 1 Adhesion, cytoskeleton regulation M22832 Krt1-18 keratin complex 1, acidic, gene  2.9 3.2  3.6 100 100 100 0.00 0.01 0.00 18 AF011450 Col15a1 procollagen, type XV  1.7 5.1   4.6 50 94 100 0.04 0.06 0.03 AA170696 Icam1 intercellular   adhesion   molecule* T  3.3 1.8  2.8 100 56 100 0.04 0.18 0.01 AW228162 Dsc2 desmocollin 2 rR  4.1 4.1  4.4 100 94 100 0.00 0.00 0.00 AI152659 Dsg2 desmoglein 2 1  3.0 3.5  3.5 100 100 100 0.01 0.02 0.00 L11316 Ect2 ect2 oncogene 10.3 4.6  4.4 100 94 100 0.02 0.12 0.20 Proteolysis, invasion, angiogenesis M84683 Muc1 mucin   1,   transmembrane* T  3.0 2.5  2.1 100 100 91 0.00 0.01 0.08 X81627 Lcn2 lipocalin 2  4.8 7.3  4.7 100 100 100 0.00 0.02 0.08 AI786089 Kng kininogen 22.4 37.1 12.6 100 100 100 0.02 0.03 0.09 AI042655 Hgfac hepatocyte growth factor  4.7 9.6  8.7 100 100 100 0.01 0.01 0.01 activator AA608277 Adora2b adenosine A2b receptor  5.8 4.1  2.4 100 75 33 0.00 0.10 0.44 AW230369 Spint1 serine protease inhibitor, Kunitz  3.0 2.7  2.3 100 100 100 0.00 0.00 0.02 type 1 AA726223 Adam19 a disintegrin and 1  3.2 3.3  3.1 100 100 100 0.00 0.02 0.02 metalloproteinase domain 19 (meltrin beta) AF030065 Hpn hepsin  5.0 4.7  5.3 100 100 100 0.00 0.00 0.00 AW047185 Thop1 thimet   oligopeptidase   1 R  4.0 2.8  4.2 100 69 100 0.00 0.01 0.02 (b) Down regulated genes in C-Myc induced lung tumors TNF-signaling M83649 Tnfrsf6 tumor   necrosis   factor   receptor R 1 −4.2 −2.6 −3.6 100 87 91 0.02 0.02 0.01 superfamily,   member   6 Y00208 Hoxa5 homeo   box   A5 R −6.1 −7.4 −7.7 100 100 100 0.00 0.00 0.00 TGF-beta-signaling AI850533 Bmp6 bone morphogenetic protein 6 1 −15.5  −34.8 −22.8  100 100 100 0.00 0.00 0.00 AJ009862 Tgfb1 Tgf-beta ? −2.9 ? −7.2 75 ? 83 0.04 0.01 AA717826 Dpt dermatopontin −5.0 −6.7 −16.6   100 100 100 0.02 0.01 0.01 L48015 Acvrl1 activin A receptor, type II-like 1 −5.9 −6.7 −7.6 100 100 100 0.00 0.00 0.00 X77952 Eng endoglin −6.7 −6.9 −7.6 100 100 100 0.00 0.00 0.00 D76432 Zfhx1a zinc finger homeobox 1a −6.8 −6.8 −8.5 100 100 100 0.00 0.00 0.00 AV138783 Gadd45b growth arrest and DNA-damage- rR 1 −1.5 −3.5 58 100 0.01 0.01 0.02 inducible 45 beta negative regulation of IGF-1-mediated survival pathway L12447 Igfbp5 insulin-like growth factor −6.5 −1.9 −4.9 100 75 100 0.00 0.25 0.00 binding protein 5 X81584 Igfbp6 insulin-like growth factor −8.8 −8.8 −18.9   100 100 100 0.00 0.00 0.00 binding protein 6 X76066 Igfbp4 insulin-like growth factor −2.8 −3.3 −5.3 100 100 100 0.03 0.03 0.01 binding protein 4 U88327 Socs2 suppressor of cytokine signaling 2 −3.7 −1.7 −2.3 100 87 100 0.01 0.05 0.04 U57524 Nfkbia nuclear factor of kappa light −3.1 −2.1 −2.8 100 93 100 0.00 0.01 0.00 chain gene enhancer in B-cells inhibitor, alpha negative regulation of Wnt/β-catenin signaling AI019193 Tcf7 transcription   factor   7,   T-cell T 1 −4.9 −5.8 −8.5 100 100 100 0.01 0.00 0.00 specific* AB023419 Sox7 SRY-box containing gene 7 −4.4 −5.8 −6.3 100 100 100 0.00 0.00 0.00 negative regulation of Ras/Raf/MEK/ERK-signaling X61940 Dusp1 dual   specificity   phosphatase   1 R 1 −3.7 −1.7 −2.3 100 93 100 0.00 0.17 0.11 D83484 Ptpre protein tyrosine phosphatase, −3.0 −4.5 −4.5 100 100 100 0.00 0.00 0.00 receptor type, E AB018194 Ptpns1 protein tyrosine phosphatase, −2.0 −3.9 −4.7 75 100 100 0.01 0.00 0.00 non-receptor type substrate 1 AF053959 Rassf5 Ras association (RalGDS/AF-6) −2.8 −3.2 −3.7 100 100 100 0.00 0.00 0.00 domain family 5 Scaffolding proteins, chaperones AB020886 Akap12 A   kinase   (PRKA)   anchor   protein R 1 −5.8 −9.0 −11.9   100 100 100 0.00 0.00 0.00 (gravin)   12 AF033275 Akap2 A kinase (PRKA) anchor rR −3.3 −3.3 −4.8 100 93 91 0.01 0.01 0.01 protein 2 AI747654 Cav caveolin,   caveolae   protein R 1 −4.0 −5.6 −6.5 100 100 100 0.00 0.00 0.00 AW120711 Dnajb9 DnaJ (Hsp40) homolog, rR 1 −3.0 −2.8 −2.1 100 100 83 0.01 0.00 0.00 subfamily B, member 9 AF040252 Fkbp7 FK506 binding protein 7 1 −3.6 −3.2 −4.1 100 100 100 0.00 0.00 0.00 Anti-angiogenic, anti-invasive AF080090 Sema3f sema domain, immunoglobulin −4.9 −5.4 −5.6 100 100 100 0.01 0.00 0.00 domain(Ig), short basic domain, secreted, (semaphorin) 3 F X70854 Fbln1 fibulin 1 !!! rR −3.8 −3.1 −10.1   100 100 100 0.00 0.00 0.00 D21165 Vsnl1 visinin-like 1 −7.1 −7.1 −7.1 100 87 100 0.00 0.00 0.00 AB006960 Reck reversion-inducing-cysteine-rich 1 −4.1 −5.9 −5.2 100 100 100 0.00 0.00 0.00 protein with kazal motifs D38117 Capn2 calpain 2 −3.0 −3.0 −3.8 100 100 100 0.02 0.03 0.02 Adhesion, ECM AI853217 Cdh5 cadherin 5 −7.2 −9.9 −10.0  100 100 100 0.00 0.00 0.00 AI844853 Spock2 sparc/osteonectin, cwcv and −6.6 −11.8 −22.3   100 100 100 0.00 0.00 0.00 kazal-like domains proteoglycan 2 X14432 Thbd thrombomodulin 1 −16.0  −27.1 −55.8   100 100 100 0.00 0.00 0.00 X80764 Tie1 tyrosine kinase receptor 1 −8.4 −12.7 −12.2  100 100 100 0.00 0.00 0.00 X65493 Icam2 intercellular adhesion molecule 2 −12.0  −30.6 −48.1   100 100 100 0.00 0.00 0.00 D13664 Osf2- osteoblast   specific   factor   2 R −3.7 −3.5 −5.4 100 100 100 0.00 0.00 0.00 pending (fasciclin   I-like) X71426 Tek endothelial-specific receptor −12.6  −16.9 −26.3   100 100 100 0.00 0.00 0.00 tyrosine kinase AW061260 Nes nestin −3.1 −4.3 −4.9 100 100 100 0.00 0.00 0.00 AA754887 Cd97 CD97   antigen* T −8.4 −9.5 −9.7 100 100 100 0.00 0.00 0.00 AI843063 Vwf Von Willebrand factor homolog −8.4 −7.7 −16.9   100 100 100 0.00 0.00 0.00 AA880988 Xlkd1 extra cellular link domain- −12.2  −11.7 −28.2   100 100 100 0.02 0.02 0.02 containing 1 U66166 Sparcl1 SPARC-like 1 (mast9, hevin) −2.9 −3.2 −5.4 100 100 100 0.00 0.00 0.00 M60474 Marcks myristoylated alanine rich −5.8 −4.1 −5.0 100 93 91 0.02 0.01 0.01 protein kinase C substrate AI854794 Tenc1 tensin like C1 domain- −5.6 −5.1 −6.8 100 100 100 0.03 0.03 0.02 containing phosphatase L23769 Mfap2 microfibrillar-associated protein rT −5.2 −5.5 −5.8 100 100 100 0.01 0.00 0.01 2* U30292 Col13a1 procollagen, type XIII, alpha 1 rR −6.2 −7.3 −16.7   100 100 100 0.01 0.01 0.01 D86917 Pcdha6 protocadherin alpha 6 −7.6 −8.6 −9.0 100 100 100 0.00 0.00 0.00 U69176 Lama4 laminin, alpha 4 −3.0 −3.1 −2.9 100 100 100 0.00 0.00 0.00 X84014 Lama3 laminin, alpha 3 −3.5 −4.8 −5.2 100 100 100 0.01 0.00 0.01 AF041409 Itga8 integrin alpha 8* rT −7.5 −10.6 −8.8 100 100 100 0.02 0.02 0.02 D86916 Pcdha4 protocadherin alpha 4 −9.9 −9.9 −9.9 100 100 100 0.00 0.00 0.00 X75285 Fbln2 fibulin 2 R −3.7 −3.3 −2.2 100 100 75 0.00 0.00 0.00 M77123 Vtn vitronectin −6.6 −6.6 −6.6 100 93 100 0.00 0.00 0.00 U12884 Vcam1 vascular cell adhesion molecule 1 1 −4.7 −3.7 −4.1 100 93 91 0.01 0.01 0.01 X79199 Tna tetranectin (plasminogen −11.1  −11.1 −11.1  100 100 100 0.00 0.00 0.00 binding protein) AW060556 Stab1 stabilin 1 −3.0 −3.3 −3.4 100 100 100 0.01 0.00 0.01 Cell junctions U82758 Cldn5 claudin 5 −9.1 −17.8 −45.9   100 100 100 0.00 0.00 0.00 Growth suppressors Z38110 Pmp22 peripheral   myelin   protein −6.2 −9.6 −16.3   100 100 100 0.00 0.00 0.00 X58289 Ptprb protein tyrosine phosphatase, −14.3  −21.3 −40.9   100 100 100 0.00 0.00 0.00 receptor type, B L09562 Ptprg protein tyrosine phosphatase, −5.7 −3.5 −4.0 100 87 91 0.01 0.02 0.01 receptor type, G AF099973 Slfn2 schlafen2 ? −3.3 ? −3.7 75 ? 91 0.03 0.03 AV349686 Ndr2 N-myc downstream regulated 2 −5.8 −4.4 −4.8 100 100 100 0.00 0.00 0.00 AI882555 Ets1 E26 avian leukemia oncogene 1, −4.2 −6.1 −5.3 100 100 100 0.01 0.00 0.00 5′ domain D10837 Lox lysyl   oxidase R 1 −3.8 −2.3 −4.9 100 87 100 0.02 0.03 0.01 D11374 Sipa1 signal-induced proliferation −3.0 −3.3 −3.9 100 100 100 0.00 0.00 0.00 associated gene 1 D76440 Ndn necdin −7.6 −8.4 −10.4  100 100 100 0.05 0.05 0.04 AF030001 Notch4 Notch   gene   homolog   4 T −5.7 −6.2 −5.8 100 100 100 0.02 0.02 0.03 ( Drosophila )*   ? X74134 Nr2f1 nuclear   receptor   subfamily   2, R −3.1 −3.1 −3.1 100 87 100 0.00 0.00 0.00 group   F,   member   1 Transformation-associated decrease Z16406 Meox2 mesenchyme homeobox 2 −8.6 −12.6 −12.6  100 100 100 0.03 0.03 0.02 X60367 Rbp1 retinol binding protein 1, cellular 2 −3.3 −1.3 −1.5 100 75 66 0.01 0.72 0.56 AI152789 Sema7a sema domain, immunoglobulin −3.9 −4.5 −7.5 100 87 83 0.03 0.02 0.02 domain (Ig), and GPI membrane anchor, (semaphorin) 7A X85994 Sema3c sema domain, immunoglobulin 1 −5.1 −4.6 −2.5 100 93 91 0.01 0.01 0.04 domain (Ig), short basic domain, secreted, (semaphorin) 3C Z80941 Sema3e sema domain, immunoglobulin −2.5 −4.2 −3.5 100 100 100 0.00 0.00 0.01 domain (Ig), short basic domain, secreted, (semaphorin) 3E Z68618 Tagln transgelin −3.9 −4.5 −4.9 100 100 100 0.00 0.00 0.00 AI841606 Ablim1 actin-binding LIM protein 1 −4.4 −5.6 −6.8 100 100 100 0.01 0.01 0.01 T- known c-Myc-target R- known c-Myc responsive gene rT- relative of known c-Myc-target rR- relative of known c-Myc-responsive gene N- number of potential c-Myc binding sites in promotor sequence *% Increase/Decrease - concordance of change calls in the pairwise comparisons (each tumor to each normal lung sample), by which genes are up or down regulated respectively Bold gene cell indicates that gene was not detected in all control lungs (for up regulated genes). Bold row indicates that gene was not detected in all control lungs and was expressed in all tumor samples (for up regulated genes) and inversely for down regulated genes.

TABLE 7 Fold changes of gene expression in lung tumors determined by RT-PCR and microarray analysis Mean FC size of tumors Gene Method small middle large Ros1 RT-PCR 3.48 3.11 3.32 Affymetrix 9.26 13.22 21.85 Ect2 RT-PCR 3.55 2.53 3.11 Affymetrix 10.25 4.61 4.44 Traf4 RT-PCR 2.27 2.19 2.06 Affymetrix 4.30 5.02 4.49 Hgfac RT-PCR 3.98 6.28 6.50 Affymetrix 4.70 9.57 8.65 Adam19 RT-PCR 2.72 2.58 2.07 Affymetrix 3.17 3.33 3.14 Bmp6 RT-PCR −9.27 −28.44 −32.45 Affymetrix −15.47 −34.84 −22.81 Acvrl1 RT-PCR −3.65 −7.11 −8.34 Affymetrix −5.89 −6.73 −7.59 Igfbp5 RT-PCR −2.90 −1.92 −2.41 Affymetrix −6.51 −1.94 −4.85 Sema3f RT-PCR −2.11 −2.33 −2.90 Affymetrix −4.90 −5.40 −5.60 Cav RT-PCR −1.90 −2.96 −2.76 Affymetrix −4.04 −5.64 −6.54 Cdh5 RT-PCR −2.74 −3.80 −3.80 Affymetrix −7.18 −9.90 −9.95

TABLE 8 Similar and different gene expression changes in c-Myc - induced bronchioalveolar carcinoma (BAC) and papillary lung adenocarcinoma (PLAC). Representative BAC, F BAC, M BAC, F P, I/D BAC, M P, I/D BAC, F BAC, M PLAC PLAC P, I/D Public ID Gene Title Gene Symbol FC FC (%) (%) T-test T-test FC (%) PLAC T-test 1. Genes induced/repressed in BAC and PLAC 1a. stronger induced/repressed in PLAC than in BAC BC006728 myelocytomatosis oncogene Myc 5.02 3.18 100 100 Up Up 42.23 100 Up NM_007629 cyclin B1, related sequence 1 /// cyclin B1 Ccnb1 2.82 3.05 100 88 Up Up 14.27 85 Up NM_007659 cell division cycle 2 homolog A (S. pombe) Cdc2a 2.97 2.96 100 100 Up Up 12.79 60 Up NM_007900 ect2 oncogene Ect2 3.53 2.19 100 94 Up Up 6.25 97 Up AF237702 serine hydroxymethyl transferase 1 Shmt1 2.84 1.79 100 50 Up Up 7.98 100 Up (soluble) AK011784 insulin-like growth factor binding protein 2 Igfbp2 −2.72 −2.08 100 100 Down Down −12.03 100 Down X82786 antigen identified by monoclonal antibody Mki67 1.83 2.1 94 100 Up Up 13.14 80 Up Ki 67 AW061324 cell division cycle 20 homolog (S. cerevisiae) Cdc20 2.12 1.82 100 69 Up Up 5.87 95 Up X66032 cyclin B2 Ccnb2 1.71 1.58 100 75 Up Up 5.56 75 Up U80932 serine/threonine kinase 6 Aurka 1.69 1.52 75 75 Up Up 4.52 80 Up AB013819 baculoviral IAP repeat-containing 5 Birc5 1.62 1.85 88 94 Up Up 6.18 70 Up AA681998 CDC28 protein kinase regulatory subunit 2 Cks2 1.77 1.23 94 63 Up None 7.15 100 Up L23755 solute carrier family 19 (sodium/hydrogen Slc19a1 1.56 1.27 100 50 Up None 9.07 90 Up exchanger), member 1 U01915 topoisomerase (DNA) II alpha Top2a 2.24 2.21 100 100 Up Up 7.24 60 Up AW122030 phosphoserine aminotransferase 1 Psat1 1.7 1.34 100 63 Up None 5.95 100 Up 1b. equally or stronger induced in BAC than in PLAC NM_008591 met proto-oncogene Met 11.99 5.82 100 75 Up None 6.16 60 Up NM_010207 fibroblast growth factor receptor 2 Fgfr2 2.75 2.16 100 75 Up None 1.79 90 Up X66083 CD44 antigen Cd44 3.6 2.14 100 75 Up None 3.28 85 Up BI788645 syndecan 1 Sdc1 3.63 2.2 100 75 Up None 2.79 100 Up NM_018874 pancreatic lipase related protein 1 Pnliprp1 8.4 5.9 100 88 Up Up 4.36 42 Up BI665568 SMC4 structural maintenance of Smc4l1 3.34 3.11 100 81 Up Up 1.75 72 Up chromosomes 4-like 1 (yeast) BG067031 NMDA receptor-regulated gene 1 Narg1 2.68 1.89 100 75 Up Up 1.89 77 Up BG069505 solute carrier family 12, member 2 Slc12a2 3.25 2.67 100 81 Up Up 1.69 92 Up BB269715 hypoxia inducible factor 1, alpha subunit Hif1a 5.11 5.39 88 63 Up None 1.51 70 Up NM_012010 eukaryotic translation initiation factor 2, Eif2s3x 4.71 3.22 100 75 Up None 1.67 72 Up subunit 3, structural gene X-linked AI314055 trans-golgi network protein Tgoln1 4.49 4.17 100 75 Up None 1.84 87 Up X66091 splicing factor, arginine/serine-rich 1 Sfrs1 3.53 2.47 100 75 Up None 1.59 67 Up (ASF/SF2) BF136532 Protein phosphatase 2, regulatory subunit Ppp2r5c 3.31 3.01 100 75 Up None 1.87 82 Up B (B56), gamma isoform BB758819 ribonucleotide reductase M1 Rrm1 3.47 1.91 100 75 Up None 2.95 50 Up M63801 gap junction membrane channel protein Gja1 4.09 2.9 100 75 Up None 2.24 92 Up alpha 1 BG073415 neural precursor cell expressed, Nedd4 3.7 2.76 100 81 Up None 1.7 87 Up developmentally down-regulted gene 4 BC010204 forty-two-three domain containing 1 Fyttd1 2.57 1.79 100 75 Up None 2.06 87 Up AV045658 ring finger protein 4 Rnf4 3.42 2.65 100 75 Up None 2.16 90 Up 1c. increased in BAC AF124142 thymoma viral proto-oncogene 3 Akt3 2.97 1.88 94 69 Up None —′ —′ —′ NM_009665 S-adenosylmethionine decarboxylase 1 /// Amd1 /// Amd2 3.13 2.18 100 81 Up None —′ —′ —′ S-adenosylmethionine decarboxylase 2 BI410363 adducin 3 (gamma) Add3 3.78 2.6 100 88 Up Up —′ —′ —′ BG069699 dCMP deaminase Dctd 3.85 2.31 94 81 Up Up —′ —′ —′ BE915516 ELL associated factor 1 Eaf1 4.48 2.55 100 69 Up None —′ —′ —′ NM_023209 PDZ binding kinase Pbk 3.39 3.15 100 88 Up Up —′ —′ —′ AF335583 platelet-derived growth factor, D Pdgfd 2.74 1.91 100 75 Up None —′ —′ —′ polypeptide BE629162 transmembrane protein 23 Tmem23 2.74 1.74 94 69 Up None —′ —′ —′ AU045529 budding uninhibited by benzimidazoles 1 Bub1b 2.82 1.81 94 25 Up None —′ —′ —′ homolog, beta (S. cerevisiae) BE533039 stress 70 protein chaperone, microsome- Stch 3.02 2.28 100 75 Up None —′ —′ —′ associated, human homolog BB749708 tousled-like kinase 1 Tlk1 2.95 2.28 100 69 Up None —′ —′ —′ NM_011803 Kruppel-like factor 6 Klf6 3.55 2.63 100 81 Up Up —′ —′ —′ 2. Genes with different regulation in BAC and PLAC 2.1 significantly induced in BAC and not regulated/repressed in PLAC NM_009163 sphingosine phosphate lyase 1 Sgpl1 6.59 4.6 100 75 Up None −1.21 37 None NM_021356 growth factor receptor bound protein 2- Gab1 2.71 1.72 94 75 Up None −1.16 42 None associated protein 1 BI662324 guanine nucleotide binding protein, alpha Gna13 3.35 2.67 100 88 Up Up −1.3 60 Down 13 BF302166 guanine nucleotide binding protein, alpha Gna12 2.78 1.85 94 75 Up None −1.78 100 Down 12 M81483 protein phosphatase 3, catalytic subunit, Ppp3cb 3 1.81 100 81 Up Up −1.24 0 None beta isoform C76813 a disintegrin and metalloproteinase Adam17 2.74 2.18 100 75 Up None 1.34 0 None domain 17 NM_009535 Yamaguchi sarcoma viral (v-yes) oncogene Yes1 5.28 2.68 100 75 Up None −1.1 0 None homolog 1 BI134907 catenin (cadherin associated protein), beta Ctnnb1 2.51 1.85 100 81 Up None 1.29 37 Up 1, 88 kDa U36502 signal transducer and activator of Stat5a 4.32 2.32 100 56 Up None 1.32 5 Up transcription 5A M33324 growth hormone receptor Ghr 2.62 2.43 100 75 Up None −2.46 100 Down BE981338 GPI-anchored membrane protein 1 Gpiap1 5.19 4.22 100 75 Up None 1.29 57 Up BB810450 transferrin receptor Tfrc 5.89 5.7 100 75 Up None 1.18 12 None AF173681 thioredoxin interacting protein Txnip 2.91 2.2 100 81 Up None 1.16 32 None BM213828 karyopherin (importin) alpha 3 Kpna3 2.72 2.32 100 75 Up None 1.13 32 None AW544889 karyopherin (importin) beta 1 Kpnb1 3.22 2.78 100 75 Up None 1.24 32 Up BG066916 ethanolamine kinase 1 Etnk1 5.88 4.68 100 81 Up None 1 12 None AV118744 apoptosis inhibitor 5 Api5 3.77 2.75 100 75 Up None 1.08 7 None BF134200 Baculoviral IAP repeat-containing 4 Birc4 3.58 2.79 100 75 Up Up −1.34 5 None NM_008173 nuclear receptor subfamily 3, group C, Nr3c1 7.29 4.93 100 75 Up Up −1.86 85 Down member 1 NM_018824 solute carrier family 23 (nucleobase Slc23a2 4.09 2.61 94 56 Up None −1.95 90 Down transporters), member 2 NM_026228 solute carrier family 39 (metal ion Slc39a8 4.36 2.51 100 75 Up None −4.1 100 Down transporter), member 8 NM_007646 CD38 antigen Cd38 3.29 2.39 100 81 Up Up −3.68 90 Down BB623587 integrin alpha 8 Itga8 3.31 2.58 100 75 Up None −9.72 100 Down X83506 utrophin Utrn 4.11 2.77 100 75 Up None −2.86 100 Down NM_009502 vinculin Vcl 6.14 3.88 100 75 Up None −2.4 100 Down NM_009640 angiopoietin 1 Angpt1 13.73 6.03 100 75 Up None −4.65 100 Down AB015595 calcitonin receptor-like Calcrl 6.08 3.78 100 75 Up None −26.4 100 Down NM_010101 endothelial differentiation, sphingolipid G- Edg3 10.76 3.48 100 75 Up None −1.99 72 Down protein-coupled receptor, 3 AF039601 transforming growth factor, beta receptor Tgfbr3 4.32 3.03 94 63 Up None −3.93 92 Down III NM_008056 frizzled homolog 6 (Drosophila) Fzd6 4.39 2.95 100 81 Up Up 1.42 0 None BE995678 tumor rejection antigen gp96 Tra1 2.51 1.63 100 75 Up None 1.4 52 Up AK003821 anaphase-promoting complex subunit 5 Anapc5 2.66 2.03 100 81 Up Up 1.37 65 Up NM_011926 CEA-related cell adhesion molecule 1 /// Ceacam1 /// Ceacam2 6.06 3.64 88 75 Up None 1.23 40 None CEA-related cell adhesion molecule 2 AK021111 serine/threonine kinase 3 (Ste20, yeast Stk3 3.29 2.1 100 81 Up Up −1.32 17 None homolog) AA124924 RAS p21 protein activator 1 Rasa1 4.57 2.5 100 75 Up Up −1.03 12 None AB015978 oncostatin M receptor Osmr 3.69 1.65 100 69 Up Up 1.04 30 None M93954 tissue inhibitor of metalloproteinase 2 Timp2 4.55 3.34 100 81 Up None −2.84 100 Down AB018194 protein tyrosine phosphatase, non- Ptpns1 2.61 1.56 94 75 Up None −3.19 92 Down receptor type substrate 1 BB400304 toll interacting protein Tollip 3.74 2.69 100 75 Up None −1.32 62 Down NM_013657 sema domain, immunoglobulin domain Sema3c 2.72 2.68 100 75 Up None −3.74 95 Down (Ig), short basic domain, secreted, (semaphorin) 3C 2.2 significantly induced/repressed in PLAC, not in BAC AB025409 CDC28 protein kinase 1 Cks1/Cks1b 1.3 1.28 63 44 None Up 7.48 100 Up M33934 inosine 5′-phosphate dehydrogenase 2 Impdh2 1.32 1.13 75 31 Up None 4.09 100 Up AI131895 kinesin family member 22 Kif22 2.41 2.12 13 25 Up Up 5.99 87 Up AI846534 NIMA (never in mitosis gene a)-related Nek6 1.22 −1.02 6 0 Up None 3.25 100 Up expressed kinase 6 AI850362 uridine-cytidine kinase 2 Uck2 1.01 −1.36 0 6 None None 3.78 100 Up AI042655 hepatocyte growth factor activator Hgfac −1.07 −1.24 0 6 None None 7.83 100 Up Z67748 spermidine synthase Srm 1.2 −1.06 31 6 Up None 4.33 95 Up Y11666 hexokinase 2 Hk2 −1.06 −1.3 13 38 None Down 3.47 100 Up L41352 amphiregulin Areg −1.83 −1.44 63 63 None None 7.1 90 Up AA726223 a disintegrin and metalloproteinase Adam19 1.14 −1.11 6 6 None None 3.23 100 Up domain 19 (meltrin beta) AW121294 ras homolog gene family, member U Arhu/Rhou −1.07 −1.06 6 13 None None 4 67 Up U51805 arginase 1, liver Arg1 1.54 1.84 6 6 None None 10.58 97 Up U15443 Ros1 proto-oncogene Ros1 −1.73 1.23 0 6 None None 14.64 90 Up M17516 lactate dehydrogenase 1, A chain Ldh1 1.1 1.02 6 0 None None 3.48 100 Up AF030065 hepsin Hpn −1.07 −1.07 0 6 None None 4.96 100 Up AV109962 Tnf receptor associated factor 4 Traf4 −1.18 1.19 0 0 None None 4.64 100 Up AI786089 kininogen Kng1 Absent Absent 0 0 25.34 100 Up AI853173 RNA polymerase 1-3 Rpo1-3 1.04 −1.04 0 0 None None 2.94 92 Up X13135 fatty acid synthase Fasn 1.18 −1.02 50 0 Up None 3.02 100 Up AW121447 nucleolar protein 5A Nol5a 1.25 1.13 25 38 None None 4.66 92 Up AW047185 thimet oligopeptidase 1 Thop1 1.1 −1.06 13 0 None None 3.58 87 Up X81627 lipocalin 2 Lcn2 −1.41 −1.45 56 56 None None 5.76 100 Up X65128 growth arrest specific 1 Gas1 1.23 1.26 0 0 Up None −6.15 100 Down X61452 septin 4 Sept4 −1.34 −1.14 75 19 Down None −3.02 100 Down AI850533 bone morphogenetic protein 6 Bmp6 −1.18 −1.1 19 13 None None −22.71 100 Down AF035207 BCL2/adenovirus E1B 19 kDa-interacting Bnip2 −1.16 −1.01 38 0 None None −2.9 100 Down protein 1, NIP2 AB020886 A kinase (PRKA) anchor protein (gravin) Akap12 −1.19 1.01 0 0 None None −8.23 100 Down 12 Z31664 activin A receptor, type II-like 1 Acvrl1 −1.05 −1.09 13 19 None None −5.54 100 Down AI747654 caveolin, caveolae protein Cav/Cav1 −1.08 −1.02 0 0 None None −5.24 100 Down D38117 calpain 2 Capn2 −1.02 −1.11 0 6 None None −3.21 100 Down X67083 DNA-damage inducible transcript 3 Ddit3 −1.66 −1.21 88 44 Down Down −3.38 100 Down AJ243895 hairy/enhancer-of-split related with Hey1 −1.09 −1.11 6 0 None Down −4.35 97 Down YRPW motif 1 X81584 insulin-like growth factor binding protein 6 Igfbp6 −1.15 −1.16 13 13 Down None −10.5 100 Down AI849416 large tumor suppressor 2 Lats2 1.8 2.08 0 0 None None −3.77 100 Down D10837 lysyl oxidase Lox 2.04 1.24 0 0 Up None −3.15 95 Down AI844911 protein tyrosine phosphatase, receptor Ptprd −1.18 1.09 25 6 None None −5.64 100 Down type, D AF080090 sema domain, immunoglobulin domain Sema3f −1.19 −1.26 44 63 None None −5.3 100 Down (Ig), short basic domain, secreted, (semaphorin) 3 F AF010254 serine (or cysteine) proteinase inhibitor, Serping1 −1.06 −1.1 0 6 None None −5.02 100 Down clade G, member 1 FC (fold change) - the ratio between mean gene expression levels in the c-Myc transgenic lungs and control non-transgenic animals P, I/D (%) - number (%) of “Present” and “Increase”/“Decrease” calls in the pairwise comparisons, each tumor sample versus each control lung sample, by which genes were up or down regulated respectively T-test - the significance of gene expression changes acoordingly to T-test (p-value cut-off 0.05) M—males, F—females For analysis of gene expression changes in BAC 4 RNA pools from c-Myc - transgenic lungs were compared with 4 RNA pools from control lungs, each pool combined RNA isolated from 4 different mice, for each gender separately.. For analysis of gene expression changes in PLAC 10 tumor nodes from different mice were compared with 4 lungs of control non-transgenic animals. 

1. A method for at least one of diagnosing cancer, prognosing cancer, staging cancer, and monitoring the treatment of cancer, comprising: (a) measuring the level of at least one biomarker selected from the group consisting of Satb1, Hspa9a, Hey1, Gas1, Bnip2, Capn2, Anp32a, Ddit3, Ccnb2, Cdkn2d (p19), Prc1, Uck2, Srm, Shmt1, Slc19a1, Npm1, Npm3, Nol5, Lamr1/Rpsa, Arhu(Rhou), Traf4, Adam19, Bmp6, Rbp1, Reck, Ect2 in a patient or in a biological sample from a patient suffering from or susceptible to cancer, and (b) comparing the level of said at least one biomarker in said patient or in said sample to a reference level of said at least one biomarker, wherein an elevated or depressed level of the biomarker relative to the reference level is indicative in at least one of diagnosing cancer, prognosing cancer, staging cancer, and monitoring the treatment of cancer.
 2. The method of claim 1, comprising monitoring the therapeutic treatment of a patient suffering from lung cancer.
 3. The method of claim 1 wherein said at least one biomarker is selected from the group consisting of Ccnb2, Slc19a1, Uck2, Srm1, Nol5a, Arhu, Adam19, Ect2, Shmt1, Gas1, Bmp6, Bnip2, Capn2, Ddit3, and Hey1.
 4. The method of in claim 3, wherein said at least one of diagnosis, prognosis, and treatment monitoring is for papillary lung adenocarcinoma (PLAC).
 5. The method of claim 2, wherein said treatment is selected from at least one member of the group consisting of treatment with atirinotecan, treatment with paclitaxel, treatment with 5-fluorouracil, and treatment with a drug binding to an epithelial cell adhesion molecule.
 6. The method of claim 1, wherein said prognosis includes differentiating between different subtypes of lung cancer.
 8. A method for the treatment of lung carcinoma, comprising administering to a patient in need of treatment a composition selected from the group consisting of: (a) a composition that decreases the expression or activity of a c-myc modulated gene selected from the group consisting of Prc1, Klt4, Ect2, Cdc20, Stk6, Nek6, Ect2, Birc5, Hspa9a, Cideb, Pglyrp, Zfp239, Elf5, Arg1, Hk1, Gapd, Suclg2, Tpi, Gnpnat1, Pign, Uck2, Gapd, Mre11a, Top2a, Ard1, Hmgb2, Xrcc5, Rrm1, Rrm2, Smarcc1, Npm3, Nol5, Lamr1, H1fx, Lmnb1, Spnr, Npm3, Nola1, Mki67ip, Ppan, Rnac, Grwd1, Srr, Pycs, Pcbd, Mrps5, Lamr1, Mrp112, Rp144, Eif2b, Tomm40, Slc15a2, Slc4a7, Slc4a4, Rangnrf, Kpnb3, Ipo4, Mlp, Stk39, Rbp1, Reck, Areg, Ros1, Arhu, Frat2, Traf4, Myc, Frat2, Cldn2, Gjb3, Gja1, Krt1-18, Col15a1, Dsg2, Ect2, Lcn2, Kng, Hgfac, Adora2b, Spint1, Adam19, and Hpn; and (b) a composition that increases the expression or activity of a c-myc modulated gene selected from the group consisting of Satb1, Cdkn2d, Lats2, Hey1, Stat1, Bnip2, Capn2, Anp32a, Madh6, Foxf1a, Tbx3, Tcf21, Gata3, Sox2, Crap, Trim30, Klf7, Sox17, Sox18, Meis1, Foxf2, Anp32a, Bmp6, Tgfb1, Dpt, Acvrl1, Eng, Zfhx1a, Igfbp5, Igfbp6, Igfbp4, Socs2, Nfkbia, Sox7, Ptpre, Ptpns1, Rassf5, Fkbp7, Sema3f, Vsnl1, Reck, Capn2, Cdh5, Spock2, Thbd, Tie1, Icam2, Tek, Nes, Vwf, Xlkd1, Sparcl1, Marcks, Tenc1, Pcdha6, Lama4, Lama3, Pcdha4, Vtn, Vcam1, Tna, Stab1, Cldn5, Pmp22, Ptprb, Ptprg, Slfn2, Ndr2, Ets1, Sipa1, Ndn, Meox2, Rbp1, Sema7a, Sema3c, Sema3e, Tagln, and Ablim1.
 9. A primer pair selected from the group consisting of Ccnb1, fp: (SEQ ID NO 3) CAGTTGTGTGCCCAAGAAGA; rp: (SEQ ID NO 4) TCCATTCACCGTTGTCAAGA; Cdc2a,, fp: (SEQ ID NO 5) CTCGGCTCGTTACTCCACTCGAGCATCAAGAAAGAGGTCAAAGG; rp: (SEQ ID NO 6) CCATTTTGCCAGAGATTCGT; Stk6: fp: (SEQ ID NO 7) GCCCACTAGGAAAAGGGAAG. rp: (SEQ ID NO 8) CGTTTGCCAACTCAGTGATG; Cdk4, fp: (SEQ ID NO 9) AACTGATCGGGACATCAAGG, rp: (SEQ ID NO 10) CACGGGTGTTGCGTATGTA; Nek6, fp: (SEQ ID NO 11) TTGAGATGATGGATGCCAAA, rp: (SEQ ID NO 12) AGCTGTGATGAACACGTTGG; Prc1, fp: (SEQ ID NO 13) CATGATGCCGAGATTGTACG, rp: (SEQ ID NO 14) CAGCCGATGTAATTCCCACT; Birc5, fp: (SEQ ID NO 15) GAATCCTGCGTTTGAGTCGT, rp: (SEQ ID NO 16) CAGGGGAGTGCTTTCTATGC; Ddit3, fp: (SEQ ID NO 17) CTGCCTTTCACCTTGGAGAC, rp: (SEQ ID NO 18) GGGCACTGACCACTCTGTTT; Satb1 fp: (SEQ ID NO 19) GTGATGGCTCAGTTGCTGAA, rp: (SEQ ID NO 20) CATAGCCCGAAGGTTTACCA; Hey1 fp: (SEQ ID NO 21) GAGACCATCGAGGTGGAAAA, rp: (SEQ ID NO 22) ACCCCAAACTCCGATAGTCC (58° C., 32 cycles); and β-actin fp: (SEQ ID NO 23) GGCATTGTTACCAACTGGGACG, rp: (SEQ ID NO 24) CTCTTTGATGTCACGCACGATTTC.


10. A method for preparing a medicament, comprising providing a composition that has a function selected from the group consisting of (a) decreasing the expression or activity of a c-myc modulated gene selected from the group consisting of Prc1, Klt4, Ect2, Cdc20, Stk6, Nek6, Ect2, Birc5, Hspa9a, Cideb Pglyrp, Zfp239, Elf5, Uck2, Smarcc1, Arg1, Hk1, Gapd, Suclg2, Tpi, Gnpnat1, Pign, Gapd, Mre11a, Top2a, Ard1, Hmgb2, Xrcc5, Rrm1, Rrm2, Smarcc1, Npm3, Nol5, Lamr1, H1fx, Lmnb1, Spnr, Npm3, Nola1, Mki67ip, Ppan, Rnac, Grwd1, Srr, Pycs, Pcbd, Mrps5, Lamr1, Mrp112, Rp144, Eif2b, Tomm40, Slc15a2, Slc4a7, Slc4a4, Rangnrf, Kpnb3, Ipo4, Mlp, Stk39, Rbp1, Reck, Areg, Ros1, Arhu, Frat2, Traf4, Myc, Frat2, Cldn2, Gjb3, Gja1, Krt1-18, Col15a1, Dsg2, Ect2, Lcn2, Kng, Hgfac, Adora2b, Spint1, Adam19, and Hpn; and (b) increasing the expression or activity of a c-myc modulated gene selected from the group consisting of Cdkn2d, Lats2, Hey1, Stat1, Bnip2, Capn2, Anp32a, Madh6, Foxf1a, Tbx3, Tcf21, Gata3, Sox2, Crap, Trim30, Klf7, Sox17, Sox18, Meis1, Foxf2, Satb1, Anp32a, Bmp6, Tgfb1, Dpt, Acvrl1, Eng, Zfhx1a, Igfbp5, Igfbp6, Igfbp4, Socs2, Nfkbia, Sox7, Ptpre, Ptpns1, Rassf5, Fkbp7, Sema3f, Vsnl1, Reck, Capn2, Cdh5, Spock2, Thbd, Tie1, Icam2, Tek, Nes, Vwf, Xlkd1, Sparcl1, Marcks, Tenc1, Pcdha6, Lama4, Lama3, Pcdha4, Vtn, Vcam1, Tna, Stab1, Cldn5, Pmp22, Ptprb, Ptprg, Slfn2, Ndr2, Ets1, Sipa1, Ndn, Meox2, Rbp1, Sema7a, Sema3c, Sema3e, Tagln, and Ablim1.
 11. The method of claim 8, wherein said medicament prevents, treats, or ameliorates the symptoms of papillary adenocarcinoma of the lung.
 12. The method of claim 8, further comprising screening for and to identifying drugs against lung carcinoma.
 13. The method of claim 10, further comprising the steps of: (c) contacting a test compound with a polypeptide encoded by the selected gene; (d) detecting the binding activity between the polypeptide and the test compound; and (e) selecting a compound that binds to the polypeptide.
 14. The method of claim 10, further, comprising the steps of: (c) contacting a test compound with a polypeptide encoded by the selected gene; (d) detecting the biological activity of the polypeptide of step (c); and (e) selecting a compound that performs at least one function selected from the group consisting of (i) suppressing the biological activity of the polypeptide encoded by the gene selected from the group consisting of Prc1, Klt4, Ect2, Cdc20, Stk6, Nek6, Ect2, Birc5, Hspa9a, Cideb Pglyrp, Zfp239, Elf5, Uck2, Smarcc1, Arg1, Hk1, Gapd, Suclg2, Tpi, Gnpnat1, Pign, Gapd, Mre11a, Top2a, Ard1, Hmgb2, Xrcc5, Rrm1, Rrm2, Smarcc1, Npm3, Nol5, Lamr1, H1fx, Lmnb1, Spnr, Npm3, Nola1, Mki67ip, Ppan, Rnac, Grwd1, Srr, Pycs, Pcbd, Mrps5, Lamr1, Mrp112, Rp144, Eif2b, Tomm40, Slc15a2, Slc4a7, Slc4a4, Rangnrf, Kpnb3, Ipo4, Mlp, Stk39, Rbp1, Reck, Areg, Ros1, Arhu, Frat2, Traf4, Myc, Frat2, Cldn2, Gjb3, Gja1, Krt1-18, Col15a1, Dsg2, Ect2, Lcn2, Kng, Hgfac, Adora2b, Spint1, Adam19, and Hpn in comparison with the biological activity detected in the absence of the test compound; and (ii) enhancing the biological activity of the polypeptide encoded by the polynucleotide selected from the group consisting of Cdkn2d, Lats2, Hey1, Stat1, Bnip2, Capn2, Anp32a, Madh6, Foxf1a, Tbx3, Tcf21, Gata3, Sox2, Crap, Trim30, Klf7, Sox17, Sox18, Meis1, Foxf2, Satb1, Anp32a, Bmp6, Tgfb1, Dpt, Acvrl1, Eng, Zfhx1a, Igfbp5, Igfbp6, Igfbp4, Socs2, Nfkbia, Sox7, Ptpre, Ptpns1, Rassf5, Fkbp7, Sema3f, Vsnl1, Reck, Capn2, Cdh5, Spock2, Thbd, Tie1, Icam2, Tek, Nes, Vwf, Xlkd1, Sparcl1, Marcks, Tenc1, Pcdha6, Lama4, Lama3, Pcdha4, Vtn, Vcam1, Tna, Stab1, Cldn5, Pmp22, Ptprb, Ptprg, Slfn2, Ndr2, Ets1, Sipa1, Ndn, Meox2, Rbp1, Sema7a, Sema3c, Sema3e, Tagln, Ablim1, in comparison with the biological activity detected in the absence of the test compound.
 15. The method of claim 14 wherein said biological activity is cell proliferation.
 16. The method of claim 1, further comprising contacting a biological or biotechnological system is contacted with a soluble substance having affinity with at least one of the genes selected from the group of Prc1, Klt4, Ect2, Cdc20, Stk6, Nek6, Ect2, Birc5, Hspa9a, Cideb Pglyrp, Zfp239, Elf5, Uck2, Smarcc1, Arg1, Hk1, Gapd, Suclg2, Tpi, Gnpnat1, Pign, Gapd, Mre11a, Top2a, Ard1, Hmgb2, Xrcc5, Rrm1, Rrm2, Smarcc1, Npm3, Nol5, Lamr1, H1fx, Lmnb1, Spnr, Npm3, Nola1, Mki67ip, Ppan, Rnac, Grwd1, Srr, Pycs, Pcbd, Mrps5, Lamr1, Mrp112, Rp144, Eif2b, Tomm40, Slc15a2, Slc4a7, Slc4a4, Rangnrf, Kpnb3, Ipo4, Mlp, Stk39, Rbp1, Reck, Areg, Ros1, Arhu, Frat2, Traf4, Myc, Frat2, Cldn2, Gjb3, Gja1, Krt1-18, Col15a1, Dsg2, Ect2, Lcn2, Kng, Hgfac, Adora2b, Spint1, Hpn, and/or mRNA encoded thereby and/or their gene products and/or parts thereof and wherein the soluble substance is linked with a marker.
 17. The method of claim 16, further comprising determining hybridization of a selected gene probe to a gene transcript of a biological sample.
 18. The method of claim 1, further comprising the steps of isolating a biological sample from biological material received from an organism, in particular from a human patient, suffering from lung cancer or from an organism to be tested for its susceptibility to lung cancer, and determining the level of at least one member of the group consisting of Prc1, Klt4, Ect2, Cdc20, Stk6, Nek6, Ect2, Birc5, Hspa9a, Cideb Pglyrp, Zfp239, Elf5, Uck2, Smarcc1, Arg1, Hk1, Gapd, Suclg2, Tpi, Gnpnat1, Pign, Gapd, Mre11a, Top2a, Ard1, Hmgb2, Xrcc5, Rrm1, Rrm2, Smarcc1, Npm3, Nol5, Lamr1, H1fx, Lmnb1, Spnr, Npm3, Nola1, Mki67ip, Ppan, Rnac, Grwd1, Srr, Pycs, Pcbd, Mrps5, Lamr1, Mrp112, Rp144, Eif2b, Tomm40, Slc15a2, Slc4a7, Slc4a4, Rangnrf, Kpnb3, Ipo4, Mlp, Stk39, Rbp1, Reck, Areg, Ros1, Arhu, Frat2, Traf4, Myc, Frat2, Cldn2, Gjb3, Gja1, Krt1-18, Col15a1, Dsg2, Ect2, Lcn2, Kng, Hgfac, Adora2b, Spint1, Adam19, Hpn, Cdkn2d, Lats2, Hey1, Stat1, Bnip2, Capn2, Anp32a, Madh6, Foxf1a, Tbx3, Tcf21, Gata3, Sox2, Crap, Trim30, Klf7, Sox17, Sox18, Meis1, Foxf2, Satb1, Anp32a, Bmp6, Tgfb1, Dpt, Acvrl1, Eng, Zfhx1a, Igfbp5, Igfbp6, Igfbp4, Socs2, Nfkbia, Sox7, Ptpre, Ptpns1, Rassf5, Fkbp7, Sema3f, Vsnl1, Reck, Capn2, Cdh5, Spock2, Thbd, Tie1, Icam2, Tek, Nes, Vwf, Xlkd1, Sparcl1, Marcks, Tenc1, Pcdha6, Lama4, Lama3, Pcdha4, Vtn, Vcam1, Tna, Stab1, Cldn5, Pmp22, Ptprb, Ptprg, Slfn2, Ndr2, Ets1, Sipa1, Ndn, Meox2, Rbp1, Sema7a, Sema3c, Sema3e, Tagln, Ablim1, and fragments thereof, by screening the presence of said proteins or of fragments of thereof or of mRNA coding for the same.
 19. The method of claim 18, wherein the gene expression profiling comprises the steps of synthesizing a cDNA library derived of the isolated RNA by RT-PCR; synthesizing a cRNA library, derived of the cDNA library by second strand cDNA synthesis and in vitro transcription of the double stranded cDNA; producing a RNA fragment library derived of the cRNA library by hydrolytic cleavage into RNA fragments; performing a hybridization assay by incubating an oligonucleotide array including spatially addressed solid phase bound oligonucleotide sequences coding for the at least two oligonucleotide sequences to be screened or for parts of thereof or for sequences being complementary of the same, with the cRNA fragment library; and scanning the hybridization pattern of the oligonucleotide array.
 20. A test kit for identifying and/or determining lung carcinoma comprising two or more detection reagents which bind to one or more genes selected from the group consisting of Prc1, Klt4, Ect2, Cdc20, Stk6, Nek6, Ect2, Birc5, Hspa9a, Cideb Pglyrp, Zfp239, Elf5, Uck2, Smarcc1, Arg1, Hk1, Gapd, Suclg2, Tpi, Gnpnat1, Pign, Gapd, Mre11a, Top2a, Ard1, Hmgb2, Xrcc5, Rrm1, Rrm2, Smarcc1, Npm3, Nol5, Lamr1, H1fx, Lmnb1, Spnr, Npm3, Nola1, Mki67ip, Ppan, Rnac, Grwd1, Srr, Pycs, Pcbd, Mrps5, Lamr1, Mrp112, Rp144, Eif2b, Tomm40, Slc15a2, Slc4a7, Slc4a4, Rangnrf, Kpnb3, Ipo4, Mlp, Stk39, Rbp1, Reck, Areg, Ros1, Arhu, Frat2, Traf4, Myc, Frat2, Cldn2, Gjb3, Gja1, Krt1-18, Col15a1, Dsg2, Ect2, Lcn2, Kng, Hgfac, Adora2b, Spint1, Adam19, Hpn, Cdkn2d, Lats2, Hey1, Stat1, Bnip2, Capn2, Anp32a, Madh6, Foxf1a, Tbx3, Tcf21, Gata3, Sox2, Crap, Trim30, Klf7, Sox17, Sox18, Meis1, Foxf2, Satb1, Anp32a, Bmp6, Tgfb1, Dpt, Acvrl1, Eng, Zfhx1a, Igfbp5, Igfbp6, Igfbp4, Socs2, Nfkbia, Sox7, Ptpre, Ptpns1, Rassf5, Fkbp7, Sema3f, Vsnl1, Reck, Capn2, Cdh5, Spock2, Thbd, Tie1, Icam2, Tek, Nes, Vwf, Xlkd1, Sparcl1, Marcks, Tenc1, Pcdha6, Lama4, Lama3, Pcdha4, Vtn, Vcam1, Tna, Stab1, Cldn5, Pmp22, Ptprb, Ptprg, Slfn2, Ndr2, Ets1, Sipa1, Ndn, Meox2, Rbp1, Sema7a, Sema3c, Sema3e, Tagln, Ablim1, mRNA thereof, and polypeptides encoded thereby. 